Humanized anti-CD40 antibodies and methods of administering thereof

ABSTRACT

The present disclosure relates to anti-CD40 antibodies, such as humanized anti-CD40 antibodies, that may be used in various therapeutic, prophylactic and diagnostic methods. The antibodies generally block the ability of CD40 to bind CD154 and do so without activating the cell expressing CD40 (e.g., a B cell). The present antibodies or fragments thereof may be used to reduce complications associated with organ or tissue transplantation.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/955,393 filed Apr. 17, 2018, which is a division of U.S. patentapplication Ser. No. 15/829,352 filed Dec. 1, 2017, which is acontinuation of International Patent Application No. PCT/US16/50114filed Sep. 2, 2016, which claims benefit of U.S. Provisional ApplicationNo. 62/214,411 filed Sep. 4, 2015.

SEQUENCE LISTING

This application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 26, 2018, is named11212_005211-US3_ST25.txt and is 33,416 bytes in size.

FIELD

The invention relates to humanized anti-CD40 antibodies and uses of suchantibodies, for example, to reduce the likelihood of, or treat,transplant rejection, to induce immunosuppression, or to treat anautoimmune disorder.

BACKGROUND OF THE INVENTION

Suppression of the immune system, particularly the humoral immunesystem, is beneficial in organ transplantation and treatment ofautoimmune disorders. Organ transplantation has emerged as a preferredmethod of treatment for many forms of life-threatening diseases thatinvolve organ damage. However, transplantation rejection may occur whenan organism receiving transplanted cells or tissue mounts an undesiredimmune response to that tissue. Transplant rejection may be minimized bytissue-type matching, but even matched tissue can be rejected by thedonor. Thus, immunosuppressive therapies are now used for virtually allcases of tissue transplantation.

Improved results in clinical transplantation have been achievedprimarily through the development of increasingly potent non-specificimmunosuppressive drugs to inhibit rejection responses. While short-termresults have improved, long-term outcomes remain inadequate. Life-longimmunosuppressive agents may be required to combat chronic rejection ofthe transplanted organ, and the use of these agents dramaticallyincreases the risks of cardiovascular disease, infections, andmalignancies.

One potential target for reducing transplantation rejection is theCD40/CD154 interaction. CD40 is expressed primarily on the surface of Blymphocytes and other antigen-presenting cells (APCs) such as dendriticcells and macrophages. CD154 is expressed primarily on the surface of Tcells. The interaction between these two proteins is associated with Bcell activation, which triggers cytokine expression as well asexpression of cell surface markers including CD23, CD80, and CD86, KehryM. R., CD40-mediated signaling in B cells. Balancing cell survival,growth, and death, J. Immunol. 1996; 156: 2345-2348. Blockade of thisinteraction using anti-CD154 antibodies has been shown to reduce oreliminate rejection of transplanted tissues in non-human primates.

For any type of immunosuppression (e.g., in a transplantationprocedure), a balance between efficacy and toxicity is a key factor forits clinical acceptance. Thus, there is a need for therapies thatspecifically target the immunological pathways involved in, for example,transplant rejection and autoimmune disorders.

SUMMARY

The present disclosure provides, for a humanized anti-CD40 antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion, wherein the heavy chain variable region comprises three CDRs,CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%identical to the amino acid sequences set fonts in SEQ ID NOs: 13, 14and 15, respectively.

The present disclosure also provides for a humanized anti-CD40 antibody,or an antigen-binding portion thereof, comprising a light chain variableregion, wherein the light chain variable region comprises three CDRs,CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about100%, identical to the amino acid sequences set forth in SEQ ID NOs: 16,17 and 18, respectively.

Also encompassed by the present disclosure is a humanized anti-CD40antibody, or an antigen-binding portion thereof, comprising a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises three complementarity determiningregions (CDRs), CDR1, CDR2 and CDR3 having amino acid sequences about80% to about 100% identical to the amino acid sequences set forth in SEQID NOs: 13, 14 and 15, respectively, and wherein the light chainvariable region comprises three CDRs, CDR1, CDR2 and CDR3, having aminoacid sequences about 80% to about 100% identical to the amino acidsequences set forth in SEQ ID NOs: 16, 17 and 18, respectively.

The present disclosure provides for a humanized anti-CD40 antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises an amino acid sequence about 80% to about 100%identical to any one of the amino acid sequences set forth in SEQ IDNOs. 11, 19, 20, 21, 24, 25 and 26.

The present disclosure provides for a humanized anti-CD40 antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the light chainvariable region comprises an amino acid sequence about 80% to about 100%identical to any one of the amino acid sequences set forth in SEQ IDNOs: 12, 22, 23, 27, 28 and 29.

The present disclosure provides for a humanized anti-CD40 antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises an amino acid sequence about 80% to about100%, identical to any one of the amino acid sequences set forth in SEQID NOs: 11, 19, 20, 21, 24, 25 and 26, and wherein the light chainvariable region comprises an amino acid sequence about 80% to about 100%identical to any one of the amino acid sequences set forth in SEQ IDNOs: 12, 22, 23, 27, 28 and 29.

The present disclosure provides for a humanized anti-CD40 antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises an amino acid sequence about 80% to about 100%identical to any one of the amino acid sequences set forth in SEQ IDNOs: 19, 20 and 21, and wherein the light chain variable regioncomprises an amino acid sequence about 80% to about 100% identical toeither of the amino acid sequences set forth in SEQ ID NOs: 22 and 23.

The present disclosure provides for a humanized anti-CD40 antibody or anantigen-binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises an amino acid sequence about 80% to about 100%identical to any one of the amino acid sequences set forth in SEQ IDNOs: 24, 25 and 26, and wherein the light chain variable regioncomprises an amino acid sequence about 80% to about 100% identical toany one of the amino acid sequences set forth in SEQ ID NOs: 27, 28 and29.

The present disclosure provides for a humanized anti-CD40 antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises an amino acid sequence about 80% to about 100%identical to the amino acid sequences set forth in SEQ ID NO: 21, andwherein the light chain variable region comprises an amino acid sequenceabout 80% to about 100% identical to the amino acid sequences set forthin SEQ ID NO: 23.

The dissociation constant (K_(D)) of the antibody, or antigen-bindingportion thereof, may be less than about 1×10⁻⁹ M, or less than about1×10⁻⁸ M.

The present antibody or antigen-binding portion thereof may be: (a) awhole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) anF(ab′)2; and/or (e) a disulfide linked Fv.

The present antibody or antigen-binding portion thereof may comprise atleast one constant domain selected from; a) an IgG constant domain; and(b) an IgA constant domain.

The present antibody or antigen-binding portion thereof may comprise atleast one human constant domain.

The present antibody or antigen-binding portion thereof may bind to CD40extracellular domain.

The CD40 may be human or rhesus CD40.

The present antibody or antigen-binding portion thereof may block Blymphocyte activation by CD154-expressing Jurkat cells in vitro.

The present antibody or antigen-binding portion thereof may inhibit Blymphocyte CD23, CD80, or CD86 expression.

Also encompassed by the present disclosure is a composition comprisingthe present antibody or antigen-binding portion thereof, and at leastone pharmaceutically acceptable carrier.

The present disclosure provides for a polynucleotide encoding thepresent antibody or antigen-binding portion thereof. The presentdisclosure provides for a vector comprising the present polynucleotide,and a cell comprising the vector.

The present disclosure provides for an isolated polypeptide comprisingthe present antibody or antigen-binding portion thereof.

Also encompassed by the present disclosure is a method of producing thepresent antibody or antigen-binding portion thereof. The method maycomprise the following steps: (a) culturing the present cells in culturemedium under conditions wherein the polynucleotide encoding the presentantibody or antigen-binding portion thereof is expressed, therebyproducing at least one polypeptide comprising the antibody orantigen-binding portion thereof; and (b) recovering the polypeptide fromthe cells or culture medium.

The present disclosure also provides for a method of suppressing theimmune system in a subject, comprising the step of administering to thesubject an effective amount of the present antibody or antigen-bindingportion thereof.

The present disclosure provides for a method of treating or treatingprophylactically transplant rejection, or increasing the duration oftime before transplant rejection occurs, in a subject in need thereof,the method comprising the step of administering to the subject aneffective amount of the present antibody or antigen-binding portionthereof.

The present disclosure provides for a method of treating or treatingprophylactically graft-versus-host disease, in a subject in needthereof, the method comprising the step of administering to the subjectan effective amount of the present antibody or antigen-binding portionthereof.

The present disclosure provides for a method of treating or treatingprophylactically an autoimmune disorder in a subject in need thereof,the method comprising the step of administering to the subject aneffective amount of the present antibody or antigen-binding portionthereof.

The subject may have received, or is in need of, an organtransplantation, and/or a tissue transplantation. The organ may be aheart, kidney, lung, liver, pancreas, intestine, and thymus, or aportion thereof. The tissue may be bone, tendon, cornea, skin, heartvalve, vein, or bone marrow.

The subject may be a human or a mammal.

The administration may be commenced prior to the transplantation. Theadministration may continue for at least one month following thetransplantation. The administration may continue for at least six monthsfollowing the transplantation of the graft.

The autoimmune disorder may be associated with or caused by the presenceof an autoantibody.

The autoimmune disorder may be systemic lupus erythematosus (SLE), CRESTsyndrome (calcinosis, Raynaud's syndrome, esophageal dysmotility,sclerodactyl, and telangiectasia), opsoclonus, inflammatory myopathy(e.g., polymyositis, dermatomyositis, and inclusion-body myositis),systemic scleroderma, primary biliary cirrhosis, celiac disease (e.g.,gluten sensitive enteropathy), dermatitis herpetiformis, Miller-FisherSyndrome, acute motor axonal neuropathy (AMAN), multifocal motorneuropathy with conduction block, autoimmune hepatitis, antiphospholipidsyndrome, Wegener's granulomatosis, microscopic polyangiitis,Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmunehepatitis, scleromyositis, myasthenia gravis, Lambert-Eaton myasthenicsyndrome, Hashimoto's thyroiditis, Graves' disease, Paraneoplasticcerebellar degeneration, Stiff person syndrome, limbic encephalitis.Isaacs Syndrome, Sydenham's chorea, pediatric autoimmuneneuropsychiatric disease associated with Streptococcus (PANDAS),encephalitis, diabetes mellitus type 1, and/or Neuromyelitis optica.

The autoimmune disorder may be pernicious anemia, Addison's disease,psoriasis, inflammatory bowel disease, psoriatic arthritis, Sjögren'ssyndrome, lupus erythematosus (e.g., discoid lupus erythematosus,drug-induced lupus erythematosus, and neonatal lupus erythematosus),multiple sclerosis, and/or reactive arthritis.

The autoimmune disorder may be polymyositis, dermatomyositis, multipleendocrine failure, Schmidt's syndrome, autoimmune uveitis, adrenalins,thyroidites, autoimmune thyroid disease, gastric atrophy, chronichepatis, lupoid hepatitis, atherosclerosis, presenile dementia,demyelinating diseases, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, alopecia arcata, pemphigoid, scleroderma,progressive systemic sclerosis, adult onset diabetes mellitus (e.g.,type II diabetes), male and female autoimmune infertility, ankylosingspondolytis, ulcerative colitis, Crohn's disease, mixed connectivetissue disease, polyarteritis nedosa, systemic necrotizing vasculitis,juvenile onset rheumatoid arthritis, glomerulonephritis, atopicdermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease,sarcoidosis, rheumatic fever, asthma, recurrent abortion,anti-phospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic activehepatitis, bird-fancier's lung, allergic disease, allergicencephalomyelitis, toxic epidermal necrolysis, alopecia, Alport'ssyndrome, alveolitis, allergic alveolitis, fibrosing alveolitis,interstitial lung disease, erythema nodosum, pyoderma gangrenosum,transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis,Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis,schistosomiasis, giant cell arteritis, ascariasis, aspergillosis,Sampler's syndrome, eczema, lymphomatoid granulomatosis, Behcet'sdisease, Caplan's syndrome, Kawasaki's disease, dengue, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic facitis, Shulman's syndrome,Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochroniccyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura,graft versus host disease, transplantation rejection, humanimmunodeficiency virus infection, echovirus infection, cardiomyopathyAlzheimer's disease, parvovirus infection, rubella virus infection, postvaccination syndromes, congenital rubella infection, Hodgkin's andnon-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma,Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma,cryoglobulinemia, Waldenstrom's macroglobuierma, Epstein-Barr virusinfection, mumps, Evan's syndrome, and/or autoimmune gonadal failure.

The administration may be parenteral, intravenous, subcutaneous,intramuscular, transdermal, oral, topical, intrathecal, or local.

The present method may further comprise administration of animmunosuppressant within six months of the administration of the presentantibody or antigen-binding portion thereof.

The immunosuppressant may be a calcineurin inhibitor, tacrolimus, anmTor inhibitor, fingolimod, myriocin, alemtuzumab, rituximab, ananti-CD4 monoclonal antibody, an anti-LFA1 monoclonal antibody, ananti-LFA3 monoclonal antibody, an anti-CD45 antibody, an anti-CD19antibody, monabetacept, belatacept, indolyl-ASC; azathioprine,lymphocyte immune globulin and anti-thymocyte globulin [equine],mycophenotate mofetil, mycophenolate sodium, daclizumab, basiliximab,cyclophosphamide, prednisone, prednisolone, leflunomide, FK778, FK779,15-deoxyspergualin, busulfan, fludarabine, methotrexate,6-mercaptopurine, 15-deoxyspergualin, LF15-0195, bredinin, brequinar,and/or muromonab-CD3. The calcineurin inhibitor may be cyclosporin A orcyclosporine B. The mTor inhibitor may be sirolimus, temsirolimus,zotarolimus, or everolimus. The anti-CD45 antibody may be an anti-CD45RBantibody. In one embodiment, the immunosuppressant is belatacept.

The present antibody or antigen-binding portion thereof and theimmunosuppressant may be administered within one month, or within oneweek, of each other.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the variable regions from the heavy chain and the lightchain of the 2C10 antibody. The nucleotide sequence shown for the heavychain (SEQ ID NO: 1) includes a signal peptide (nucleotides 1-57;underlined) and the heavy chain variable sequence (nucleotides 58-396).The corresponding amino acid sequence is shown below (SEQ ID NO: 2),where amino acids 1-19 corresponding to the signal sequence (underlined)and amino acids 20-132 correspond to the heavy chain variable region.

The nucleotide sequence shown for the light chain (SEQ ID NO: 3)includes a signal peptide (nucleotides 1-66; underlined) and the lightchain variable sequence (nucleotides 67-384). The corresponding aminoacid sequence is shown below (SEQ ID NO: 4), where amino acids 1-22correspond to the signal peptide (underlined) and amino acids 23-128correspond to the light chain variable region.

FIG. 2a is a plot showing flow cytometry data confirming the binding of2C10 to human and rhesus CD20+ B cells.

FIG. 2b is a plot showing CD40 adsorption data from ELISA assays with avarying concentrations of 2C10 to confirm the binding of 2C10 to humanand rhesus CD40 as detected using goat anti-mouse IgG-HRP.

FIG. 3 is a graph showing the dose-dependent inhibition of CD154 bindingto human B cells by 2C10. B cells were analyzed for CD154 binding byincubating with histidine-tagged soluble CD154 and analyzing forhistidine expression. Results are representative of multiple repetitionsof the experiment.

FIG. 4 is a schematic diagram and graphs showing the principle of theassay involving rhesus or human peripheral blood mononuclear cells(PBMCs) and Jurkat cells.

FIG. 5 is a set of graphs showing CD23 expression in CD20⁺ cells takenfrom co-cultures of rhesus PBMCs and Jurkat cells in the presence ofvariable concentrations of 3A8, 5C8, or 2C10 antibodies.

FIG. 6 is a set of graphs showing CD86 expression in CD20⁺ cells takenfrom co-cultures of human PBMCs and Jurkat cells in the presence ofvariable concentrations of 3A8, 5C8, or 2C10 antibodies.

FIG. 7 is a set of graphs showing CD23 expression CD20′ cells fronteither human or rhesus PSMCs cultured without Jutkat cells in thepresence of cither the 3A8 or the 2C10 antibody.

FIG. 8 is a graph showing peripheral B cell count of rhesus macaquestreated with mouse-rhesus chimeric forms of 2C10 engineered to containeither rhesus IgG1 (2C10R1) or IgG4 I2C10R4) heavy chain constantregions, and chimeric IgG1 forms of anti-CD40 3A8 (3A8R1) or anti-CD40Chi220 (Chi220).

FIG. 9 is a graph showing T cell-dependent antibody responses in macaquemonkeys treated with 2C10R1, 2C10R4, or 3A8R1 antibody. All animals wereimmunized with 4-hydroxy-3-nitrophenylacetyl-conjugated keyhole limpethemocyanin (KLH) after the first antibody treatment.

FIG. 10 is a diagram showing the standard macaque model of allogeneicislet transplantation. Diabetes was induced in macaque monkeys usingstreptozotocin. Diabetic monkeys were transplanted with allogeneicislets and immunosuppression initiated with basiliximab and sirolumus.Experimental animals received 2C10R4 treatment on days 0 and 7post-transplantation.

FIG. 11a is a plot showing free blood glucose levels (FBG) in 4 macaquesfollowing islet transplantation, background immunosuppression, andtreatment with 2C10R4. The solid line on the plot represents the levelof 2C10 in the plasma.

FIG. 11b is a plot showing FBG in macaques that received only backgroundimmunosuppression.

FIG. 12 is a graph showing results from a competitive blockade assayusing human PBMCs incubated with increasing concentrations of 2C10, 3A8,or Chi220 antibodies and stained with an APC-conjugated 2C10 to assessthe ability of each antibody to cross-block 2C10.

FIGS. 13a and 13b show sequence alignment of humanized 2C10 variableregions. FIG. 13a : Murine 2C10 VH sequence was aligned against humangermline VH1-3 and three humanized sequences 2C10_h1, 2C10_h2, and2C10_h3. FIG. 13b : Murine 2C10 VL sequence was aligned against humangermline VH3-11 and two humanized sequences 2C10_11 and 2C10_12. The2C10 CDRs are bolded. The murine residues in humanized sequences areunderlined.

FIG. 14 shows amino acid changes in framework 3 between 2C10HP and2C10HB1, as well as 2C10HB2 constructs.

FIG. 15 shows the sequences of heavy chain and light chain variableregions for humanized 2C10 antibodies. The heavy chain and light chainvariable regions include 2C10HP, 2C10HB1, 2C10HB2, 2C10KP, 2C10KB1, and2C10KB2.

FIG. 16 shows binding affinity of humanized 2C10 antibody to CD40 fromdifferent primate species. Humanized 2C10 antibodies (h2C10) wereimmobilized to the surface of CM5 chip by amine coupling. Differentconcentrations of CD40-MBP fusions of human, rhesus, and baboon wereanalyzed for affinity on a BIACore 3000. The binding affinity wascalculated with BIAevaluation software version 4.1.1.

FIG. 17 shows that induction of anti-KLH antibody response (IgM and IgG)was determined in monkeys immunized with KLH 3 hrs after receivingeither saline, 10 or 25 mg/kg h2C10. All control animals exhibited IgGor IgM antibody responses to the KLH antigen. Individual monkeys treatedwith 10 mg/kg, but no animals treated with 25 mg/kg 2C10 developedeither an IgG or IgM antibody responses to KLH.

FIG. 18. Whole blood was used for phenotyping B and T lymphocyte subsetsafter treatment with 25 mg/kg h2C10 (Top row) or 10 mg/kg h2C10 (Middlerow), or control animals (Bottom Row). Neither dose of h2C10 had anyapparent effect on the lymphocyte populations. The absence ofappreciable B cell depletion was also evident in the earlier,dose-response evaluation of the primate chimeric form that included adetailed analysis of mature and immature B cell populations.

FIG. 19. Day 28 Humanized 2C10 fully saturated CD40 binding sites on Bcells. H2C10 administered in vivo completely blocked the binding offluorescently labeled 2C10 binding to B cells. Data illustrate resultsfrom humanized 2C10-treated and control monkeys 28 days after a singledose of 25 mg/kg (top row), 10 mg/kg (middle row) or 0 mg/kg (control,bottom row). Similar results were obtained on Days 3, 7, 14, and 21 postinfusion.

FIG. 20. Mean serum concentrations of h2C10 for up to 28 days aftertreatment of monkeys with either 10 or 25 mg/kg. Concentrations of 2C10slowly decline over time, and levels are detected over the entireduration of the study.

FIGS. 21a and 21b show the DNA and amino acid sequences of the humanized2C10 (h2C10) in the stabilized IgG4 format. FIG. 21a shows the DNA andamino acid sequences of the heavy chain. FIG. 21b shows the DNA andamino acid sequences of the light chain SEQ ID NO: 32: DNA sequence ofthe heavy chain; SEQ ID NO: 33; amino acid sequence of the heavy chain,SEQ ID NO: 34; DNA sequence of the light chain; SEQ ID NO: 35; aminoacid sequence of the light chain.

DETAILED DESCRIPTION

The present disclosure relates to anti-CD40 antibodies and antibodyfragments (e.g., antigen-binding portions of the antibody) that may beused in various therapeutic, prophylactic, diagnostic and other methods.The antibodies can block the ability of CD40 to bind CD154 and do sowithout activating the cell expressing CD40 (e.g., a B cell). Thepresent antibodies or fragments thereof may be used so reducecomplications associated with organ or tissue transplantation.

The antibodies, or antigen-binding portions thereof, include, but arenot limited to, humanized antibodies, human antibodies, monoclonalantibodies, chimeric antibodies, polyclonal antibodies, recombinantlyexpressed antibodies, as well as antigen-binding portions of theforegoing. An antigen-binding portion of an antibody may include aportion of an antibody that specifically binds to CD40.

The present disclosure also provides for compositions and methods forreducing the likelihood of transplant rejection, treat transplantrejection, inducing immunosupression, and/or treating an autoimmunedisorder. The compositions contain antibodies or fragments thereof thatspecifically bind CD40.

In one embodiment, the present disclosure provides for a method oftreating or ameliorating graft-versus-host disease and/or transplantrejection in a subject comprising administering to the mammal acomposition comprising an antibody of the invention (or its fragment) inan amount sufficient to decrease one or more of the symptoms ofgraft-versus-host disease and/or transplant rejection in the subject.

In another embodiment, the antibody or antigen-binding fragment isadministered to a subject having an inflammatory disease or an immunedisorder such as an autoimmune disease. The inflammatory disease orautoimmune disease may be associated with CD40-expressing cells.

The invention features methods of reducing the likelihood of transplantrejection, treat transplant rejection, inducing immunosuppression,and/or treating an autoimmune disorder in a subject by administering tothe subject the present antibody or antigen-binding portion thereof inan effective amount.

Also encompassed by the present disclosure is a method, of blocking thefunction of CD40 in a mammal comprising administering to the mammal acomposition comprising the present antibodies, or antigen-bindingportions thereof, in an amount sufficient to block a CD40-mediatedimmune response in the mammal.

Another method of the disclosure relates to inhibiting the growth and/ordifferentiation of cells expressing CD40, comprising administering thepresent antibody or antigen-binding fragment to the cells, wherein thebinding of the antibody or antigen-binding fragment to CD40 inhibits thegrowth and/or differentiation of the cells.

The present disclosure provides for a method of treating a subjecthaving a CD40-associated disorder, comprising administering to thesubject the present antibody or antigen-binding fragment, wherein thebinding of the antibody or antigen-binding fragment to CD40 inhibits thegrowth and or differentiation of cells of the CD40-associated disorder.The cells may be, but are not limited to, B lymphoblastoid cells,pancreatic, lung cells, breast cells, ovarian cells, colon cells,prostate cells, skin cells, head and neck cells, bladder cells, bonecells or kidney cells.

The present method may be used to treat chronic lymphocytic leukemia,Burkitt's lymphoma, multiple myeloma, a T cell lymphoma, Non-Hodgkin'sLymphoma, Hodgkin's Disease, Waldenstrom's macroglobulmemia or Kaposi'ssarcoma.

Additional methods of the present disclosure include inhibiting antibodyproduction by B cells in a subject comprising administering to thesubject, an effective amount of an anti-CD40 antibody or its fragment ofthe present disclosure. In one embodiment, the antibody is administeredin an amount effective to inhibit B cell differentiation and antibodyisotype switching in the subject. In another embodiment, the antibody isadministered in an amount effective to inhibit cytokine and chemokineproduction, and/or inhibit up-regulation of adhesion molecules inT-cells and macrophages in the subject. In a third embodiment, theantibody is administered in an amount effective to inhibit activation ofdendritic cells in the subject.

In addition to the present antibody or its fragment, the present methodsmay further comprise administering a second therapeutic agent such as animmunosuppressant, a tumor necrosis factor antagonist (aTNF-antagonist), a CTLA4-antagonist, an anti-IL-6 receptor antibody, ananti-CD20 antibody, or a combination thereof.

The present antibodies, or antigen-binding portions thereof, mayspecifically bind to human CD40 and/or rhesus CD40, includingrecombinant and native human CD40.

As used herein, a cell that expresses CD40 is any cell characterized bythe surface expression of CD40, including, but not limited to, normaland neoplastic B cells, interdigitating cells, basal epithelial cells,carcinoma cells, macrophages, endothelial cells, follicular dendriticcells, tonsil cells, and bone marrow-derived plasma cells.

Humanized Antibodies

The humanized antibody of the present disclosure is an antibody from anon-human species where the amino acid sequences in the non-antigenbinding regions (and/or the antigen-binding regions) have been alteredso that the antibody more closely resembles a human antibody, and stillretains its original binding ability.

An antibody light or heavy chain variable region consists of threehypervariable regions, referred to as complementarity determiningregions (CDRs). CDRs are supported within the variable regions byframework regions (FRs). In one embodiment, the heavy chain variableregion (or light chain variable region) contains three CDRs and fourframework regions (FRs), arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Kabat, E. A., et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services.NIH Publication No. 91-3242, 1991. Chothia, C. el al., J. Mol. Biol.196:901-917, 1987.

In certain embodiments, humanized antibodies are antibody molecules fromnon-human species having one, two, three or all CDRs from the non-humanspecies, and one, two, three, four or all framework regions from a humanimmunoglobulin molecule.

The CDRs of the present antibodies or antigen-binding portions thereofcan be from a non-human or human source. The framework of the presentantibodies or antigen-binding portions thereof can be human, humanized,non-human (e.g., a murine framework modified to decrease antigenicity inhumans), or a synthetic framework (e.g., a consensus sequence). In oneembodiment, the present antibodies, or antigen-binding portions thereofcontain at least one heavy chain variable region and/or at least onelight chain variable region.

The humanized antibodies of the present disclosure can be produced bymethods known in the art. For example, a humanized antibody can have oneor more amino acid residues introduced into it from a source which isnon-human. These non-human amino acid residues are often referred to as“import” residues, which are typically taken from an “import” variabledomain. Humanization can be performed following the method of Winter andco-workers (Jones et. al., Nature 321:522-5, 1986; Riechmann et. al.,Nature 332:323-7, 1988; Verhoeyen et al., Science 239:1534-6, 1988), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, in such humanized antibodiessubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Incertain embodiments, humanized antibodies are human antibodies in whichat least some hypervariable region residues as well as other variableregion residues are substituted by residues from analogous sites innon-human antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies, may reduce antigenicity. Accordingto the “best-fit” method, the sequence of the variable domain of anon-human (e.g., rodent such as mouse) antibody is screened against theentire library of known human variable domain sequences, the humansequence which is closest to that of the non-human is then accepted asthe human framework for the humanized antibody. See. e.g., Sims et al.,J. Immunol. 151:2296-308, 1993; Chothia et. al., J. Mol. Biol.196:901-17, 1987. Another method uses a particular framework derivedfrom the consensus sequence of all human antibodies of a particularsubgroup of light or heavy chains. The same framework may be used forseveral different humanized antibodies. See, e.g., Carter et al., Proc.Natl. Acad. Sci. USA 89:42855-9, 1992; Presta et al., J. Immunol.151:2623-32, 1993.

Humanized antibodies can be generated by replacing sequences of thevariable region that are not directly involved in antigen binding withequivalent sequences from human variable regions. Those methods includeisolating, manipulating and expressing the nucleic acid sequences thatencode all or part of variable regions from at least one of a heavy orlight chain. Sources of such nucleic acid are well known to thoseskilled in the art and, for example, may be obtained from a hybridomaproducing an antibody against CD40. The recombinant DNA encoding thehumanized antibody, or fragment thereof, can then be cloned into anappropriate expression vector.

In another example, once non-human (e.g., murine) antibodies areobtained, variable regions can be sequenced, and the location of theCDRs and framework residues determined. Kabat, E. A., et. al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242.Chothia, C. et. al. (1987) J. Mol. Biol., 196:901-917. The light andheavy chain variable regions can, optionally, be ligated tocorresponding constant regions. CDR-grafted antibody molecules can beproduced by CDR-grafting or CDR substitution. One, two, three or allCDRs of an immunoglobulin chain can be replaced. For example, all of theCDRs of a particular antibody may be from at least a portion of anon-human animal (e.g., mouse such as CDRs shown in Table 1) or onlysome of the CDRs may be replaced. It is only necessary to keep the CDRsrequired for binding of the antibody to a predetermined antigen (e.g.,CD40). Morrison, S. L., 1985, Science, 229:1202-1207. Oi et al., 1986,BioTechniques, 4:214. U.S. Pat. Nos. 5,585,089, 5,225,539, 5,693,761 and5,693,762. EP 519596. Jones et al. 1986, Nature, 321:552-525. Verhoeyanet. al., 1988, Science, 239:1534. Beidler et al., 1988, J. Immunol.,141:4053-4060.

It may be desirable that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences find variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic such as increased affinity for the target antigen(s), isachieved.

In some embodiments, a humanized anti-CD40 antibody also includes atleast a portion of an immunoglobulin constant region, typically that ofa human immunoglobulin. In one embodiment, the antibody will containboth the light chain as well as at least the variable domain of a heavychain. The antibody also may include one or more of the constant domainCH1, hinge, CH2, CH3, and/or CH4 of the heavy chain, as appropriate.

In some aspects of the present disclosure, one or more domains of thehumanized antibodies will be recombinantly expressed. Such recombinantexpression may employ one or more control sequences, i.e.,polynucleotide sequences necessary for expression of an operably linkedcoding sequence in a particular host organism. The control sequencessuitable for use in prokaryotic cells include, for example, promoter,operator, and ribosome binding site sequences. Eukaryotic controlsequences include, but are not limited to, promoters, polyadenylationsignals, and enhancers. These control sequences can be utilized forexpression and production of humanized anti-CD40 antibody in prokaryoticand eukaryotic host cells.

Also encompassed by the present disclosure are antibodies, orantigen-binding portions thereof, containing one, two, or all CDRs asdisclosed herein, with the other regions replaced by sequences from atleast one different species including, but not limited to, human,rabbits, sheep, dogs, cats, cows, horses, goats, pigs, monkeys, apes,gorillas, chimpanzees, ducks, geese, chickens, amphibians, reptiles andother animals.

Human Antibodies

Human antibodies of the disclosure can be constructed by combining Fvclone variable domain sequencers selected from human-derived phagedisplay libraries with known human constant domain sequencer(s)(Hoogenboom et al., J. Mol. Biol 227:381-8, 1992; Marks et al., J. Mol.Biol 222:581-97, 1991). Alternatively, human antibodies can be made bythe hybridoma method. Human myeloma and mouse-human heteromyeloma celllines for the production of human monoclonal antibodies have beendescribed, for example, by Kozbor, J. Immunol. 133:3001-5, 1984, Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York. 1987): and Boerner et al., J.Immunol. 147:86-95, 1991.

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining legion (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See. e.g. Jakobovits et al., Proc.Natl. Acad. Sci. USA 90:2551-5, 1993: Jakobovits et al., Nature362:255-8, 1993; Brüggemann et al., Year Immunol. 7:33-40, 1993.

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g., rodent, antibodies, where the human antibody hassimilar affinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting,”either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab where the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e., the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT Publication WO 93/06213). Unlike traditionalhumanization of non-human antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no FR or CDR residuesof non-human origin.

Chimeric Antibodies

A chimeric antibody is a molecule in which different portions arederived from different animal species. For example, an antibody maycontain a variable region derived from a murine antibody and a humanimmunoglobulin constant region. Chimeric antibodies can be produced byrecombinant DNA techniques. Morrison, et al., Proc Natl Acad Sci,81:6851-6855 (1984). For example, a gene encoding a murine (or otherspecies) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.Chimeric antibodies can also be created by recombinant DNA techniqueswhere DNA encoding murine V regions can be ligated to DNA encoding thehuman constant regions. Better et al., Science. 1988, 240:1041-1043, Liuet al. PNAS, 1987 84:3439-3443. Liu et al., J. Immunol., 1987,139:3521-3526. Sun et al. PNAS, 1987, 84:214-218. Nishimura et al.,Canc. Res., 1987, 47:999-1005. Wood et al. Nature, 1985, 314:446-449.Shaw et al., J. Natl. Cancer Inst., 1988, 80:1553-1559. InternationalPatent Publication Nos. WO1987002671 and WO 86/01533. European PatentApplication Nos. 184, 187; 171,496; 125,023; and 173,494. U.S. Pat. No.4,816,567.

Variable Regions and CDRs

The heavy chain variable regions, light chain variable regions and CDRsof the murine 2C10 antibody and certain humanized anti-CD40 antibodies,are shown in Table 1.

Chain, SEQ Name Region Sequence ID NO. 2C10 Heavy chain,QVQLQQSGAELAKPGASVKMSCKASGYTFT 11 (Murine variable regionNYWMHWVKQRPGQGLEWIGYINPSNDYTKY antibody) NQKFKDKATLTADKSSNTAYMQLGSLTSEDSAVYYCARQGFPYWGQGTLVTVSA 2C10 Light chain,QIVLTQSPAIMSASPGEKVTMTCSASSSVSYM 12 variable regionHWYHQRSGTSPKRWIYDTSKLASGVPARFSG SGSGTSYSLTISSMEAEDAATYYCHQLSSDPFTFGSGTKLEIK 2C10 Heavy chain, YTFTNYWMH 13 CDR1 2C10 Heavy chain,YINPSNDYTKYNQKFKD 14 CDR2 2C10 Heavy chain, QGFPY 15 CDR3 2C10Light chain, SASSSVSYMH 16 CDR1 2C10 Light chain, DTSKLAS 17 CDR2 2C10Light chain, HQLSSDPFT 18 CDR3 2C10_h1 Heavy chain,QVQLVQSGAEVKKPGASVKVSCKASGYTFT 19 variable regionNYWMHWVRQAPGQRLEWMGYINPSNDYTK YNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCAR QGFPYWGQGTLVTVSS 2C10_h2 Heavy chain, QVQLVQSGAEVKKPGASVKVSCKASG20 variable region YTFTNYWMH WVRQAPGQRLEWMG YINPSNDYTKYNQKFKDRVTITADKSASTAYMELSSLRSEDTAVYYCA R QGFPY WGQGTLVTVSS 2C10_h3 Heavy chain,QVQLVQSGAEVKKPGASVKVSCKASG 21 variable region YTFTNYWMH WVRQAPGQRLEWIGYINPSNDYTKYNQKFKD RATLTADKSANTAYM ELSSLRSEDTAVYYCAR QGFPY WGQGTLVTVSS2C10_l1 Light chain, EIVLTQSPATLSLSPGERATLSC SASSSVS 22 variable regionYMH WYQQKPGQAPRLLIY DTSKLAS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQLSSDPFT FGGGTKVEIK 2C10_l2 Light chain, EIVLTQSPATLSLSPGERATLSC 23variable region SASSSVSYMH WYQQKPGQAPRRWIY  DTSKLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYC HQLSSDPFT FGGGTKVEIK 2C10HPHeavy chain, QVQLVQSGAEVKKPGASVKVSCKASGYTFT 24 variable regionNYWMHWVRQAPGQRLEWIGYINPSNDYTKY NQKFKDRATLTADKSANTAYMELSSLRSEDTAVYYCARQGFPYWGQGTLVTVSS 2C10HB1 Heavy chain,QVQLVQSGAEVKKPGASVKVSCKASGYTFT 25 variable regionNYWMHWVRQAPGQRLEWIGYINPSNDYTKY NQKFKDRATLTAD T S T NTAYMELSSLRSEDTAVYYCARQGFPYWGQGTLVTVSS 2C10HB2 Heavy chain,QVQLVQSGAEVKKPGASVKVSCKASGYTFTN 26 variable regionYWMHWVRQAPGQGLEWIGYINPSNDYTKYN QKFKDKAT I TAD E S T NTAYMELSSLRSEDTAVYYCARQGFPYWGQGTLVTVSS 2C10KP Light chain,EIVLTQSPATLSLSPGERATLSCSASSSVSYMH 27 variable regionWYQQKPGQAPRRWIYDTSKLASGVPARFSGS GSGTDYTLTISSLEPEDFAVYYCHQLSSDPFTFGGGTKVEIK 2C10KB1 Light chain, DIQMTQSPSTLSASVGDRVTITCSASSSVSYM 28variable region HWYQQKPGKAPKLLIYDTSKLASGVPARFSGSGSGTEFTLTISSLQPDDFATYYCHQLSSDPFT FGQGTKVEVK 2C10KB2 Light chain,EIVLTQSPATLSLSPGERATLSCSASSSVSYMH 29 variable regionWYQQKPGQAPRLLIYDTSKLASGIPARFSGSG SGTDFTLTISSLEPEDFAVYYCHQLSSDPFTFGQGTKLEIK VH1-3 Heavy chain, QVQLVQSGAEVKKPGASVKVSCKASGYTFTS 30variable region YAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDT AVYYCARWGQGTLVTVSS VK3-11 Light chain,EIVLTQSPATLSLSPGERATLSCRASQSVSSYL 31 variable regionAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCFGGGTKVEI K

In certain embodiments the antibodies or antigen-binding portionsthereof include a heavy chain variable region comprising an amino acidsequence at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or about 100% identical to a heavychain variable region amino acid sequence as set forth in any of SEQ IDNOs: 11, 19, 20, 21, 24, 25 and 26.

In certain embodiments, the antibodies or antigen-binding portionsthereof include a light chain variable region comprising an amino acidsequence at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or about 100% identical to a lightchair, variable region amino acid sequence as set forth in any of SEQ IDNOs 12, 22, 23, 27, 28 and 29.

In certain embodiments, the antibodies or antigen-binding portionsthereof each include both a heavy chain variable region comprising anamino acid sequence at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99%, about 70%, about 75%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99% or about 100% identicalto a heavy chain variable region amino acid sequence as set forth in anyof SEQ ID NOs: 11, 19, 20, 21, 24, 25 and 26, and a light chain variableregion including an amino acid sequence at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 99%, about 70%, about 75%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% identical to a variable light chain amino acid sequence asset forth in SEQ ID NOs: 12, 22, 23, 27, 28 and 29.

A heavy chain variable region of the antibodies or antigen-bindingportions thereof can comprise one, two, three or more complementaritydetermining regions (CDRs) that are at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 70%, about 75%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% identical to the CDRs of a heavy chain variable region of the2C10 antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 13, 14,15, respectively).

A light chain variable region of the antibodies or antigen-bindingportions thereof can comprise one, two, three or more CDRs that are atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, about70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99% or about 100% identical to the CDRs of a lightchain variable region of the 2C10 antibody (CDR1, CDR2 and CDR3 as setforth in SEQ ID NOs 16, 17, 18, respectively).

A heavy chain variable region of the present antibodies, orantigen-binding portions thereof can comprise one, two, three or morecomplementarity determining regions (CDRs) that are at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 99%, about 70%, about 75%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99% or about 100% identical to the CDRs of a heavy chain variable regionof the 2C10 antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:13, 14, 15, respectively), and a light chain variable region of theantibodies or antigen-binding portions thereof can comprise one, two,three or more CDRs that are at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 99%, about 70%, about 75%, about 80%, about 81%,about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 04%,about 95%, about 96%, about 97%, about 98%, about 99% or about 100%identical to the CDRs of a light chain variable region of the 2C10antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs. 16, 17, 18,respectively.

A heavy chain variable region of the antibodies or antigen-bindingportions thereof can include three CDRs that are at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, about 70%, about 75%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% identical to the CDRs of a heavy chain variable region of the2C10 antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 13, 14,15, respectively).

In one embodiment, a light chain variable region of the antibodies orantigen-binding portions thereof includes three CDRs that are at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 99%, about 70%,about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or about 100% identical to the CDRs of a light chainvariable region of the 2C10 antibody (CDR1, CDR2 and CDR3 as set forthin SEQ ID NOs: 16, 17, 18 respectively).

In one embodiment, a heavy chain variable region of the antibodies orantigen-binding portions thereof includes three CDRs that are at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 99%, about 70%,about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 97%, about 98%, about99% or about 100% identical to the CDRs of a heavy chain variable regionof the 2C10 antibody (CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs:13, 14, 15, respectively), and a light chain variable region of theantibodies or antigen-binding portions thereof includes three CDRs thatare at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 99%,about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99% or about 100% identical to the CDRs of a lightchain variable region of the 2C10 antibody (CDR1, CDR2 and CDR3 as setforth in SEQ ID NOs: 16, 17, 18, respectively).

In certain embodiments, a heavy chain variable region of the antibodiesor antigen-binding portions thereof includes three CDRs that areidentical to CDRs of a heavy chain variable region of the 2C10 antibody(CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 13, 14, 15,respectively), and a light chain variable region of the antibodies orantigen-binding portions thereof includes three CDRs that are identicalto CDRs of a light chain variable region of the 2C10 antibody (CDR1,CDR2 and CDR3 as set forth in SEQ ID NOs: 16, 17, 18, respectively).

Encompassed by the present disclosure are antibodies with a heavy chainvariable region and a light chain variable region having amino acidsequences at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100%, identical to the heavy chainvariable region (SEQ ID NO: 11) and light chain variable region (SEQ IDNO: 12) of the antibody 2C10, respectively.

In related embodiments, anti-CD40 antibodies or antigen-binding portionsthereof include, for example, the CDRs of heavy chain variable regionsand/or light chain variable regions of 2C10.

In one embodiment, the antibody or antigen-binding portion thereofcontains a heavy chain variable region and a light chain variable regionidentical to a heavy chain variable region and light chain variableregion of the 2C10 antibody (SEQ ID NO: 11 and SEQ ID NO: 12,respectively).

In various embodiments, the antibodies or antigen-binding portionsthereof specifically bind to an epitope that overlaps with, or are atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, about70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99% or about 100%, identical to an epitope bound by the2C10 antibody. The epitope may be present within the sequence of SEQ IDNO: 6, or may be present within the sequence of amino acids 8-40 of SEQID NO: 6.

In certain embodiments, CDRs corresponding to the CDRs in Table 1 havesequence variations. For example, CDRs, in which 1, 2, 3, 4, 5, 6, 7 or8 residues, or less than 20%, less than 30%, or less than about 40%, oftotal residues in the CDR, are substituted or deleted can be present inan antibody for antigen-binding portion thereof) that binds CD40.

Also within the scope of the disclosure are antibodies orantigen-binding portions thereof in which specific amino acids have beensubstituted, deleted or added. These alternations do not have asubstantial effect on the peptide's biological properties such asbinding activity. For example, antibodies may have amino acidsubstitutions in the framework region, such as to improve binding to theantigen. In another example, a selected small number of acceptorframework residues can be replaced by the corresponding donor aminoacids. The donor framework can be a mature or germline human antibodyframework sequence or a consensus sequence. Guidance concerning how tomake phenotypically silent amino acid substitutions is provided in Bowieet al., Science, 247; 1306-1310 (1990). Cunningham et al., Science, 244;1081-1085 (1989). Ausubel (ed.), Current Protocols in Molecular Biology,John Wiley and Sons, Inc. (1994). T. Maniatis, E. F. Fritsch and J.Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harborlaboratory, Cold Spring Harbor, N.Y. (1989). Pearson, Methods Mol. Biol.243: 307-31 (1994). Gonnet et al., Science 256: 1443-45 (1992).

The present peptides may be the functionally active variant ofantibodies of antigen-binding portions thereof disclosed herein, e.g.,with less than about 30%, about 25%, about 20%, about 15%, about 10%,about 5% or about 1% amino acid residues substituted or deleted butretain essentially the same immunological properties including, but notlimited to, binding to CD40.

The antibodies or antigen-binding portions thereof may also includevariants, analogs, orthologs, homologs and derivatives of peptides, thatexhibit a biological activity, e.g., binding of an antigen such as CD40.The peptides may contain one or more analogs of an amino acid(including, for example, non-naturally occurring amino acids, aminoacids which only occur naturally in an unrelated biological system,modified amino acids from mammalian systems etc.), peptides withsubstituted linkages, as well as other modifications known in the art.

The antibody, or antigen-binding portion thereof, can be derivatized orlinked to another functional molecule. For example, an antibody can befunctionally linked (by chemical coupling, genetic fusion, noncovalentinteraction, etc.) to one or more other molecular entities, such asanother antibody, a detectable agent, an immunosuppressant, a cytotoxicagent, a pharmaceutical agent, a protein or peptide that can mediateassociation with another molecule (such as a streptavidin core region ora polyhistidine tag), amino acid linkers, signal sequences, immunogeniccarriers, or ligands useful in protein purification, such asglutathione-S-transferase, histidine tag, and staphylococcal protein A.Cytotoxic agents may include radioactive isotopes, chemotherapeuticagents, and toxins such as enzymatically active toxins of bacterial,fungal, plant, or animal origin, and fragments thereof. Such cytotoxicagents can be coupled to the humanized antibodies of the presentdisclosure using standard procedures, and used, for example, to treat apatient indicated for therapy with the antibody.

One type of derivatized protein is produced by crosslinking two or moreproteins (of the same type or of different types). Suitable crosslinkersinclude those that are heterobifunctional, having two distinct reactivegroups separated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) homobifunctional (e.g.,disuccinimidyl substrate). Useful detectable agents with which a proteincan be derivatized (or labeled) include fluorescent agents, variousenzymes, prosthetic groups, luminescent materials, bioluminescentmaterials, and radioactive materials. Non-limiting, exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, and phycoerythrin. A protein or antibody canalso be derivatized with detectable enzymes, such as alkalinephosphatase, horseradish peroxidase, beta-galactosidase,acetylcholinesterase, glucose oxidase and the like. A protein can alsobe derivatized with a prosthetic group (e.g., streptavidin/biotin andavidin/biotin).

In another embodiment, the humanized anti-CD40 antibody or its fragmentis used unlabeled and detected with a labeled antibody that binds thehumanized anti-CD40 antibody or its fragment.

Antibody Fragments

The antibodies can be full-length or can include a fragment (orfragments) of the antibody having an antigen-binding portion, including,but not limited to, Fab, F(ab′)2, Fab′, F(ab)′, Fv, single chain Fv(scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAbfragment (e.g., Ward et al., Nature, 341:544-546 (1989)), an isolatedCDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chainantibody molecules, and multispecific antibodies formed from antibodyfragments. Single chain antibodies produced by joining antibodyfragments using recombinant methods, or a synthetic linker, are alsoencompassed by The present disclosure. Bird et al. Science, 1988,242:423-426. Huston et al., Proc. Natl. Acad. Sci. USA, 1988,85:5879-5883.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

Fv is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′liagments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

Single-chain Fv or scFx antibody fragments comprise the V_(H) and V_(L)domains of antibody, where these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv, see, e.g., Pluckthün, in The Pharmacology of MonoclonalAntibodies, vol. 113, Roseuburg and Moore, eds., (Springer-Verlag, NewYork, 1904). pp 269-315

Diabodies are antibody fragments with two antigen-binding sites, whichfragments comprise a heavy-chain variable domain (V_(H)) connected to alight-chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies may be bivalent or bispecific.Diabodies are described more fully in, for example, European Patent No.404,097; PCT Publication WO 1993/01161; Hudson et al., Nat. Med.9:129-34, 2003; and Hollinger et al., Proc. Natl. Acad. Sci. USA90:6444-8, 1993. Triabodies and tetrabodies are also described in Hudsonet al., Nar. Med. 9:129-34, 2003.

Antibody fragments may be generated by traditional means, such asenzymatic digestion, or by recombinant techniques. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors. For areview of certain antibody fragments, see Hudson et al. Nat. Med.9:129-134, 2003.

Various techniques have been developed for the production of antibodyfragments. Traditionally these fragments were derived via proteolyticdigestion of intact antibodies (see e.g., Morimoto et al., J. Biochem.Biophys. Methods 24:107-17, 1992; and Brennan et al., Science 229:81-3,1985). However, these fragments can now be produced directly byrecombinant host cells. Fab, Fv, and ScFv antibody fragments can all beexpressed in and selected from E. Coli, thus allowing the facileproduction of large amounts of these fragments. Antibody fragments canbe isolated from the antibody phage libraries. Alternatively Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-7,1992). In another approach, F(ab′)₂ fragments are isolated directly fromrecombinant host cell culture. Fab and F(ab′)₂ fragment with increasedin vivo half-life comprising salvage receptor binding epitope residuesare described in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

The present antibody or antigen-binding portion thereof may comprise atleast one constant domain, such as, (a) an IgG constant domain; (b) anIgA constant domain, etc.

All antibody isotypes are encompassed by the present disclosure,including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, (IgA1, IgA2),IgD or IgE. The antibodies or antigen-building portions thereof may bemammalian (e.g., mouse, human) antibodies or antigen-binding portionsthereof. The light chains of the antibody may be of kappa or lambdatype. An alternative humanized anti-CD40 antibody can comprise sequencesfrom more than one immunoglobulin class or isotype, and selectingparticular constant domains to optimize desired effector functions iswithin the ordinary skill in the art.

The antibodies or antigen-binding portions thereof of The presentdisclosure may be monospecific, bi-specific or multi-specific.Multi-specific or bi-specific antibodies or fragments thereof may bespecific for different epitopes of one target polypeptide (e.g., CD40)or may contain antigen-binding domains specific for more than one targetpolypeptide (e.g., antigen-binding domains specific for CD40 and otherantigen relating to transplant rejection or autoimmune disease). In oneembodiment, a multispecific antibody or antigen-binding portion thereofcomprises at least two different variable domains, wherein each variabledomain is capable of specifically binding to a separate antigen or to adifferent epitope on the same antigen. Tutt et al., 1991, J. Immunol.147:60-69. Kufer et al., 2004. Trends Biotechnol 22:238-244. The presentantibodies can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present disclosure includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor CD40, and she other arm of the immunoglobulin is specific for asecond therapeutic target or is conjugated to a therapeutic moiety suchas an immunosuppressant.

Production of Antibodies

The present disclosure provides for methods for making an antibody orantigen-binding portion thereof that specifically binds to CD40.

For example, a non-human animal is immunized with a composition thatincludes CD40, and then a specific antibody is isolated from the animal.The method can further include evaluating binding of the antibody toCD40.

In one embodiment, the present disclosure provides for a method formaking a hybridoma that expresses an antibody that specifically binds toCD40. The method contains the following steps: immunizing an animal witha composition that includes CD40 or its fragment, isolating splenocytesfrom the animal, generating hybridomas from the splenocytes; andselecting a hybridoma that produces an antibody that specifically bindsto CD40. Kohler and Milstein, Nature, 256: 495, 1975. Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988.

In one embodiment, CD40 is used to immunize mice intraperitoneally orintravenously. One or more boosts may or may not be given. The titers ofthe antibodies in the plasma can be monitored by, e.g., ELISA(enzyme-linked immunosorbent assay) or flow cytometry. Mice withsufficient titers of anti-CD40 antibodies are used for fusions. Mice mayor may not be boosted with antigen 3 days before sacrifice and removalof the spleen. The mouse splenocytes are isolated and fused with PEG toa mouse myeloma cell line. The resulting hybridomas are then screenedfor the production of antigen-specific antibodies. Cells are plated, andthen incubated in selective medium. Supernatants from individual wellsare then screened by ELISA for human anti-CD40 monoclonal antibodies.The antibody secreting hybridomas are replated, screened again, and ifstill positive for anti-CD40 monoclonal antibodies, can be subcloned bylimiting dilution.

Adjuvants that may be used to increase the immunogenicity of CD40include any agent or agents that act to increase an immune response topeptides or combination of peptides. Non-limiting examples of adjuvantsinclude alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/vsqualene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitantrioleate (Span 85)). CpG-containing nucleic acid, QS21 (saponinadjuvant), MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylated MPL)extracts from Aquilla, ISCOMS (see, e.g., Sjolander et al. (1998) J.Leukocyte Biol. 64:713; WO09/03184; WO96/11711; WO 00/48630; WO98/36772;WO00/41720; WO06/134423 and WO07/026190), LT/CT mutants,poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A.,interleukins, Freund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineCGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

The immunized animal can be any animal that is capable of producingrecoverable antibodies when administered an immunogen, such as, but notlimited to, rabbits, mice, rats, hamsters, goats, horses, monkeys,baboons and humans. In one aspect, the host is transgenic and produceshuman antibodies, e.g., a mouse expressing the human immunoglobulin genesegments. U.S. Pat. No. 8,236,311; 7,625,559 and 5,770,429, thedisclosure of each of which is incorporated herein by reference in itsentirety. Lonberg et al., Nature 368(6474): 856-859, 1994. Lonberg, N.,Handbook of Experimental Pharmacology 113:49-101, 1994. Lonberg, N. andHuszar, D. Intern. Rev. Immunol. 13: 65-93, 1995 Harding, F. and LonbergN., Ann. N.Y. Acad. Sci., 764:536-546, 1995.

The present antibodies or portions thereof can be produced by host cellstransformed with DNA encoding light and heavy chains (or portionsthereof) of a desired antibody. Antibodies (or portions thereof) can beisolated and purified from these culture supernatants and/or cells usingstandard techniques. For example, a host cell may be transformed withDNA encoding the light chain, the heavy chain, or both, of an antibody.Recombinant DNA technology may also be used to remove some or all of theDNA encoding at least a portion of either or both of the light and heavychains that is not necessary for binding, e.g., the constant region.

The invention also encompasses a nucleic acid or polynucleotide encodingat least one of the present antibody or antigen-binding portion thereofthat specifically binds to CD40. The nucleic acid may be expressed in acell to produce the present antibody or antigen-binding portion thereof.The isolated nucleic acid or polynucleotide of the present disclosurecomprises at least one sequence encoding a peptide at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 99%, about 70%, about 75%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99% or about 100% identical to any of SEQ ID NOs: 11-29.

The invention also features expression vectors including at least onenucleic acid or polynucleotide encoding a peptide at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, about 70%, about 75%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% identical to any of SEQ ID NOs: 11-29.

Nucleic acid molecules encoding a functionally active variant of thepresent antibody or antigen-binding portion thereof are also encompassedby the present disclosure. These nucleic acid molecules may hybridizewith a nucleic acid encoding any of the present antibody orantigen-binding portion thereof under medium stringency, highstringency, or very high stringency conditions. Guidance for performinghybridization reactions can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which isincorporated herein by reference. Specific hybridization conditionsreferred to herein are as follows: (1) medium stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (2) high stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and (3) very high stringency hybridizationconditions; 0.5 M sodium phosphate, 7% SDS at 65° C. followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C.

A nucleic acid or polynucleotide encoding the present antibody orantigen-binding portion thereof may be introduced into an expressionvector that can be expressed in a suitable expression system, followedby isolation or purification of the expressed antibody orantigen-binding portion thereof. Optionally, a nucleic acid encoding thepresent antibody or antigen-binding portion thereof can be translated ina cell-free translation system. U.S. Pat. No. 4,816,567. Queen et al.,Proc Natl Acad Sci USA, 86; 10029-10033 (1989).

The present nucleic acids can be expressed in various suitable cells,including prokaryotic and eukaryohc cells, e.g., bacterial cells, (e.g.,E. coli), yeast cells, plant cells, insect cells, and mammalian cells. Anumber of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC). Non-limiting examples of the cells include all celllines of mammalian origin or mammalian-like characteristics, includingbut not limited to, parental cells, derivatives and/or engineeredvariants of monkey kidney cells (COS, e.g. COS-1, COS-7), HEK293, babyhamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NS0,PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2),SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphomacells. The engineered variants include, e.g., glycan profile modifiedand/or site-specific integration site derivatives.

The present disclosure also provides for cells comprising the nucleicacids described herein. The cells may be a hybridoma or transfectant.The present antibody or antigen-binding portion thereof can be expressedin various cells. The types of the cells are discussed herein.

When using recombinant techniques to produce, e.g. the humanizedantibody or the antigen-binding portion thereof, the antibody or itsportion can be produced intracellularly, in the periplasmic space, ordirectly secreted into the medium. If the antibody is producedintracellularly, the cells may be disrupted to release protein as afirst step. Particulate debris, either host cells or lysed fragments,can be removed, for example, by centrifugation or ultrafiltration.Carter et al., 1992, Bio/Technology 10:163-167 describes a procedure forisolating antibodies that are secreted to the periplasmic space of E.coli. Briefly, cell paste is thawed in the presence of sodium acetate(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30minutes. Cell debris can be removed by centrifugation. Where theantibody is secreted into the medium, supernatants from such expressionsystems may be first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A variety of methods can be used to isolate theantibody from the host cell.

The antibody or its portion prepared from the cells can be purifiedusing, for example, hydroxylapalite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a typical purification technique. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human gamma1, gamma2, orgamma4 heavy chains (see. e.g., Lindmark et al., 1983 J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and for humangamma3 (see, e.g., Guss et al, 1986 EMBO J. 5:1567-1575). A matrix towhich an affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C.sub.H3 domain, the Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Othertechniques for protein purification such as fractionation on anion-exchange column, ethanol precipitation, reverse phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation are also available depending on the antibody to berecovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elation buffer at a pHbetween about 2.5-4.5, typically performed at low salt concentrations(e.g., from about 0-0.25M salt).

Hybridomas or other cells that produce antibodies that bind, preferablywith high affinity, to CD40 can then be subcloned and furthercharacterized. One clone from each hybridoma or cell, which retains thereactivity of the parent cells (by ELISA), can then be chosen for makinga cell bank, and for antibody purification.

Alternatively, the present antibody or antigen-binding portion thereofcan be synthesized by solid phase procedures well known in the art.Solid Phase Peptide Synthesis: A Practical Approach by E. Atherton andR. C. Sheppard, published by IRL at Oxford University Press (1989)Methods in Molecular Biology, Vol 35: Peptide Synthesis Protocols (ed. MW. Pennington and B. M. Dunn), chapter 7. Solid Phase Peptide Synthesis,2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984). G. Barany and R. B.Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Grossand J. Meienhofer, Vol. 1 and Vol. 2, Academic Press, New York, (1980),pp. 3-254. M. Bodansky, Principles of Peptide Synthesis,Springer-Verlag, Berlin (1984).

Additional antibodies (e.g., monoclonal polyclonal, poly-specific, ormonospecific antibodies) against the CD40 epitope recognized by 2C10 canbe made, e.g., using a suitable method for making antibodies. In anexample, a coding sequence for an epitope recognized by the 2C10antibody is expressed as a C-terminal fusion with glutathioneS-transferase (GST) (Smith et al., Gene 67:31-40, 1988). The fusionprotein is purified on glutathione-Sepharose beads, eluted withglutathione, cleaved with thrombin (at an engineered cleavage site), andpurified for immunization of rabbits. Primary immunizations are carriedout with Freund's complete adjuvant and subsequent immunizations withFreund's incomplete adjuvant. Antibody titers are monitored by Westernblot and immunoprecipitation analyses using the thrombin-cleaved proteinfragment of the GST fusion protein. Immune sera are affinity purifiedusing CNBr-Sepharose-coupled protein. Antiserum specificity can bedetermined using a panel of unrelated GST proteins.

As an alternate or adjunct immunogen to GST fusion proteins, peptidescorresponding to relatively unique immunogenic regions of a polypeptideof the invention can be generated and coupled to keyhole limpethemocyanin (KLH) through an introduced C-terminal lysine. Antiserum toeach of these peptides is similarly affinity purified on peptidesconjugated to BSA, and specificity is tested by ELISA or Western blotanalysis using peptide conjugates, or by Western blot orimmunoprecipitation using the polypeptide expressed as a GST fusionprotein.

Alternatively, monoclonal antibodies that specifically bind the CD-40epitope recognized by the 2C10 antibody can be prepared using standardhybridoma technology (see, e.g., Kohler et al., Nature 256:495-7, 1975;Kohler et al., Eur. J. Immunol 6:511-9, 1976; Kohler et al., Eur. J.Immunol. 6:292-5, 1976, Hammerling et al., Monoclonal Antibodies and TCell Hybridomas, Elsevier, N.Y., 1981). Once produced, monoclonalantibodies can also be tested for specific recognition by Western blotor immunoprecipitation analysis. Alternatively, monoclonal antibodiescan be prepared using the polypeptide of the invention described aboveand a phage display library (Vaughan et al., Nat. Biotechnol. 14:309-14,1996).

Epitopic fragments can be generated by standard techniques, e.g., usingPCR and cloning the fragment into a pGEX expression vector. Fusionproteins are expressed in E. coli and purified using a glutathioneagarose affinity matrix. To minimize potential problems of low affinityor specificity of antisera, two or three such fusions are generated foreach protein, and each fusion is injected into at least two rabbits.Antisera are raised by injections in a series, and can include, forexample, at least three booster injections.

In order to generate polyclonal antibodies on a huge scale and at a lowcost an appropriate animal species can be chosen. Polyclonal antibodiescan be isolated from the milk or colostrum of, e.g., immunized cows.Bovine colostrum contains 28 g of IgG per liter, while bovine milkcontains 1.5 g of IgG per liter (Ontssouka et al., J. Dairy Sci. 86;2005-11, 2003). Polyclonal antibodies can also be isolated from the yolkof eggs from immunized chickens (Sarker et al., J. Pediatr.Gastroenterol. Nutr. 32:19-25, 2001).

Assays

Various methods can be used to assay the antibodies or antigen-bindingportions thereof to confirm their specificity for the antigen ofinterest and/or to study their properties. One method of conducting suchassays is a sera screen assay as described in U.S. Patent PublicationNo. 2004/0126829. Anti-CD40 antibodies can be characterized for bindingto CD40 by a variety of known techniques. For example, in an ELISA,microliter plates are coated with CD40 or a fragment of CD40 in PBS, andthen blocked with irrelevant proteins such as bovine serum albumin (BSA)diluted in PBS. Dilutions of plasma from CD40-immunized mice (orsolutions containing anti-CD40 antibodies) are added to each well andincubated. The plates are washed and then incubated with a secondaryantibody conjugated to an enzyme (e.g., alkaline phosphatase). Afterwashing, the plates are developed with the enzyme's substrate (e.g.,ABTS), and analyzed at a specific OD. In other embodiments, to determineif the selected monoclonal antibodies bind to unique epitopes, theantibody can be biotinylated which can then be detected with astreptavidin labeled probe. Anti-CD40 antibodies can be tested forreactivity with CD40 by Western blotting.

Antibodies, or antigen-binding fragments, variants or derivativesthereof of the present disclosure can also be described or specified interms of their binding affinity to an antigen. The affinity of anantibody for an antigen can be determined experimentally using anysuitable method (see, e.g., Berzofsky et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul W. E., Ed., Raven Press;New York, N. Y. (1984); Kuby, Janis Immunology, W. H. Freeman andCompany: New York, N.Y. (1992), and methods described herein. Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions (e.g., salt concentration, pH).Thus, measurements of affinity and other antigen-binding parameters(e.g., K_(D), K_(a), K_(d)) are preferably made with standardizedsolutions of antibody and antigen, and a standardized buffer.

The present antibodies or antigen-binding portions thereof specificallybind to CD40 with a dissociation constant of less than about 10⁻⁷ M,less than about 10⁻⁸ M, less than about 10⁻⁹ M, less than about 10⁻¹⁰ M,less than about 10⁻¹¹ M, less than about 10⁻¹² M, from about 10⁻⁷ M toabout 10⁻¹² M, from about 10⁻⁸ M to about 10⁻¹¹ M, from about 10⁻⁹ toabout 10⁻¹⁰ M, or from about 10⁻⁸ to about 10⁻¹² M.

Assays may also be used to test the ability of an antibody (or itsfragment) to block CD40 binding to CD154, or inhibit or decreaseCD40-mediated responses.

As used herein, the terms “inhibits binding” and “blocks binding” (e.g.,inhibition/blocking of binding of CD154 to CD40) are usedinterchangeably and encompass both partial and complete inhibitionblocking. Inhibition and blocking are also intended to include anymeasurable decrease in the binding of CD154 to CD40 when in contact withan anti-CD40 antibody or portions thereof as disclosed herein ascompared to ligand not in contact with an anti-CD40 antibody, e.g., theblocking of CD154 to CD40 by at least about 10%, 20%, 30%, 40%, 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100%.

In one embodiment, activation of B cells, or inhibition thereof, may bedetermined by measuring expression of one or more markers selected fromCD23, CD80, CD86, and any additional suitable marker on CD20+ cells.

The present antibodies or fragments thereof may be characterized bytheir effects on T cell-mediated antibody responses. For example, theantibodies or fragments thereof may inhibit IgM and/or IgG production ina mammal, when the antibody, or an antigen-binding portion thereof, isadministered to the mammal at a dosage ranging from about 1 mg/kg bodyweight to about 50 mg/kg body weight, from about 2 mg/kg body weight toabout 40 mg/kg body weight, from about 3 mg/kg body weight to about 30mg/kg body weight, from about 5 mg/kg body weight, to about 20 mg/kgbody weight, from about 8 mg/kg body weight to about 13 mg/kg bodyweight, about 1 mg/kg body weight, about 2 mg/kg body weight, about 5mg/kg body weight, about 10 mg/kg body weight, about 15 mg/kg bodyweight, about 20 mg/kg body weight, about 25 mg/kg body weight, about 30mg/kg body weight, about 35 mg/kg body weight, about 40 mg/kg bodyweight, about 50 mg/kg body weight, about 60 mg/kg body weight, about 70mg/kg body weight, or about 80 mg/kg body weight.

The present antibodies or fragments thereof may be characterized bytheir effects on prolonging graft survival post transplantation. Thepresent antibodies or fragments thereof, alone or in combination withone or more other immunosuppressants, may prolong graft survival byabout 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 2 fold, about 5 fold, about 10 fold, about 15 fold, about 20fold, about 25 fold, about 30 fold, about 35 fold, about 40 fold, about45 fold, about 50 fold, about 55 fold, greater than about 40%, greaterthan about 50%, greater than about 60%, greater than about 70%, greaterthan about 80%, or greater than about 00%, greater than about 2 fold,greater than about 5 fold, greater than about 10 fold, greater thanabout 20 fold, greater than about 30 fold, or greater than about 40fold. For example, the present antibodies or fragments thereof mayprolong islet allograft survival.

In certain embodiments, the present antibody, or antigen-bindingfragment thereof: a) may block binding of CD40 to CD154; c) may blockactivation of antigen presenting cells (e.g., B cells, dendritic cells,macrophages, etc.); d) may or may not induce depletion of B cells; e)may or may not inhibit or decrease cytokine release from antigenpresenting cells; f) may or may not induce tumor cell apoptosis, g) mayor may not inhibit tumor cell proliferation; h) may or may not killstumor cell; i) may or may not stimulate anti-tumor T cell responses;and/or j) may or may not reduce established tumors. The antibodiesdescribed herein may have or induce a combination of any one or more ofthese attributes or activities. Tai, et al., Cancer Res. 2005, 1;65(13):5898-906; Luqman et al., Blood 112:711-720, 2008. The antibodiesor portions thereof described herein may also be tested for effects onCD40 internalization, in vitro and in vivo efficacy, etc. Such assaysmay be performed using well-established protocols known to the skilledperson (see e.g., Current Protocols in Molecular Biology (Greene Publ.Assoc, Inc. & John Wiley & Sons, Inc., NY, N.Y.); Current Protocols inImmunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H.Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY,N.Y.); or commercially available kits.

Conditions to be Treated

The present antibodies or antigen-binding portions thereof have in vitroand in vivo therapeutic, prophylactic, and or diagnostic utilities. Forexample, cells can be cultured in vitro in culture medium and contactedby the anti-CD40 antibody or fragment thereof. The antibodies orantigen-binding portions thereof can be administered in a subject, aspart of an in vivo (e.g., therapeutic or prophylactic) protocol. For invivo embodiments, the contacting step is effected in a subject andincludes administering an anti-CD40 antibody or portion thereof to thesubject under conditions effective to permit binding of the antibody orportion thereof to CD40 in the subject. The antibodies orantigen-binding portions thereof can be administered to reduce thelikelihood of, or increase the duration prior to, transplant rejection,inducing immunosuppression, or treating an autoimmune disorder.

The antibodies or antibody fragments described herein may be used in anysituation in which immunosuppression is desired (e.g., transplantrejection or autoimmune disorders). These antibodies are particularlyuseful for treating transplant rejection e.g., reducing the likelihoodthat a particular transplant is rejected by the host or increasing thetime before rejection takes place. The antibodies or antibody fragmentsdescribed herein can be used in conjunction with transplantation of anyorgan or any tissue that is suitable for transplantation. Non-limitingexemplary organs include heart, kidney, lung, liver, pancreas,intestine, and thymus; non-limiting exemplary tissues include bone,tendon, cornea, skin, heart valve, vein, and bone marrow. The antibodiesand antibody fragments can also be used to treat autoimmune disorders.In one embodiment, the autoimmune disorder may be associated with orcaused by the presence of an autoantibody. Autoimmune diseases that maybe treated with the present antibodies or fragments thereof include, butare not limited to, systemic lupus erythematosus (SLE), CREST syndrome(calcinosis, Raynaud's syndrome, esophageal dysmotility, sclerodactyl,and telangiectasia), opsoclonus, inflammatory myopathy (e.g.,polymyositis, dermatomsositis, and inclusion-body myositis), systemicscleroderma, primary biliary cirrhosis, celiac disease (e.g., glutensensitive enteropathy), dermatitis herpetiformis, Miller-FisherSyndrome, acute motor axonal neuropathy (AMAN), multifocal motorneuropathy with conduction block, autoimmune hepatitis, antiphospholipidsyndrome, Wegener's granulomatosis, microscopic polyangiitis,Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmunehepatitis, scleromyositis, myasthenia gravis, Lambert-Eaton myasthenicsyndrome, Hashimoto's thyroiditis, Graves' disease, Paraneoplasticcerebellar degeneration, Stiff person syndrome, limbic encephalitis,Isaacs Syndrome, Sydenham's chorea, pediatric autoimmuneneuropsychiatric disease associated with Streptococcus (PANDAS),encephalitis, diabetes mellitus type 1, and Neuromyelitis optica. Otherautoimmune disorders include pernicious anemia, Addison's disease,psoriasis, inflammatory bowel disease, psoriatic arthritis, Sjögren'ssyndrome, lupus erythematosus (e.g., discoid lupus erythematosus,drug-induced lupus erythematosus, and neonatal lupus erythematosus),multiple sclerosis and reactive arthritis.

Additional disorders that may be heated using the methods of the presentdisclosure include, for example, polymyositis, dermatomyositis, multipleendocrine failure, Schmidt's syndrome, autoimmune uveitis, adrenalitis,thyroiditis, autoimmune thyroid disease, gastric atrophy, chronichepatitis, lupoid hepatitis, atherosclerosis, presenile dementia,demyelinating diseases, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia pemphigusvulgaris, pemphigus, alopecia areata, pemphigoid, scleroderma,progressive systemic sclerosis, adult onset diabetes mellitus (e.g.,type II diabetes), male and female autoimmune infertility, ankylosingspondolytis, ulcerative colitis, Crohn's disease, mixed connectivetissue disease, polyarteritis nedosa, systemic necrotizing vasculitis,juvenile onset rheumatoid arthritis, glomerulonephritis, atopicdermantis, atopic rhinitis, Goodpasture's syndrome, Chagas' diseasesarcoidosis, rheumatic fever, asthma, recurrent abortion,anti-phospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic activehepatitis, bird-fancier's lung, allergic disease, allergicencephalomyelitis, toxic epidermal necrolysis, alopecia, Alport'ssyndrome, alveolitis, allergic alveolitis, fibrosing alveolitis,interstitial lung disease, erythema nodosum, pyoderma gangrenosum,transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis,Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis,schistosomiasis, giant cell arteritis, ascariasis, aspergillosis,Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet'sdisease, Caplan's syndrome, Kawasaki's disease, dengue, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochroniccyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura,graft versus host disease, transplantation rejection, humanimmunodeficiency virus infection, echovirus infection, cardiomyopathy,Alzheimer's disease, parovirus infection, rubella virus infection, postvaccination syndromes, congenital rubella infection, Hodgkin's andnon-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma,Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma,cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barr virusinfection, mumps Evan's syndrome, and autoimmune gonadal failure.

In another embodiment, the present antibody or its fragment can be usedin the treatment of various disorders associated with the expression ofCD40.

A disorder may be any condition that would benefit from treatment withthe present antibody or its fragment. This includes chronic and acutedisorders or diseases including those pathological conditions thatpredispose the mammal to the disorder in question. Non-limiting examplesof disorders to be treated herein include autoimmune diseases,immunologic disorders, inflammatory disorders, cancer, hematologicalmalignancies, benign and malignant tumors, leukemia, lymphoidmalignancies, and angiogenic disorders.

As used herein, the term “CD40-associated disorder” or “CD40-associateddisease” refers to a condition in which modification or elimination ofcells expressing CD40 is indicated. These include CD40-expressing cellsdemonstrating abnormal proliferation or CD40-expressing cells that areassociated with cancerous or malignant growth. CD40-associated disordersinclude, but are not limited to, diseases and disorders of the immunesystem, such as autoimuume disorders and inflammatory disorders. Suchconditions include, but are not limited to, rheumatoid arthritis (RA),systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome,multiple sclerosis, psoriasis, inflammatory bowel disease (e.g.,ulcerative colitis and Crohn's disease, pulmonary inflammation, asthma,and idiopathic thrombocytopenic purara (ITP). More particular examplesof cancers that demonstrate abnormal expression of CD40 antigen includeB lymphoblastoid cells, Burkitt's lymphoma, multiple myeloma, T celllymphomas, Kaposi's sarcoma, osteosarcoma, epidermal and endothelialtumors, pancreatic, lung, breast, ovarian, colon, prostate, head andneck, skin (melanoma), bladder, and kidney cancers. Such disorders alsoinclude, but are not limited to, leukemias, lymphomas, including B celllymphoma and non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom'smacroglobulmemia; solid tumors, including sarcomas, such asosteosarcoma, Ewing's sarcoma, malignant melanoma, adenocarcinoma,including ovarian adenocarcinoma, Kaposi's sarcoma/Kaposi's tumor andsquamous cell carcinoma. U.S. Pat. No. 9,090,696.

CD40-expressing cancers that can be treated or prevented by the presentantibodies or fragments thereof also include, for example, leukemia,such as acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocyticor erythroleukemia), chronic leukemia, chronic myelocytic (granulocytic)leukemia, or chronic lymphocytic leukemia; Polycythemia vera; Lymphoma(e.g., Hodgkin's disease or Non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia; heavy chain disease: solid tumors suchsarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenoearcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, or esophageal carcinoma).

Also encompassed are methods of treating disorders of B lymphocytes(e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoidarthritis, and type I diabetes), of Th1-lymphocytes (e.g., rheumatoidarthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome,Hashimoto's thyroiditis, Graves's disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or of Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease).

In some embodiments, the immunological disorder is a T cell-mediatedimmunological disorder, such as a T cell disorder in which activated Tcells associated with the disorder express CD40. Anti-CD40 antibodies oragents can be administered to deplete such CD40-expressing activated Tcells. In a specific embodiment, administration of anti-CD40 antibodiesor agents can deplete CD40-expressing activated T cells, while resting Tcells are not substantially depleted by the anti-CD40 or agent. In thiscontext, “not substantially depleted” means that less than about 60%, orless than about 70% or less than about 80% of resting T cells are notdepleted.

Combination Therapy

The present antibody or antigen-binding portion thereof can beadministered alone or in combination with one or more other therapeuticagents (e.g., a second therapeutic agent). In some embodiments, thepharmaceutical compositions comprising the anti-CD40 antibody or itsfragment can further comprise a second therapeutic agent, eitherconjugated or unconjugated to the antibody or its fragment. In oneembodiment, the second agent is another monoclonal or polyclonalantibody or antigen-binding portion thereof. In another embodiment, thesecond agent is an immunosuppressant. In a third embodiment, the secondagent is a cytotoxic or cytostatic agent. In a fourth embodiment, thesecond agent may target a receptor or receptor complex other than CD40on the surface of activated lymphocytes, dendritic cells orCD40-expressing cancer cells.

Such combination therapy can have an additive or synergistic effect oncondition parameters (e.g., severity of a symptom, the number ofsymptoms, or frequency of relapse).

The present anti-CD40 antibody or its fragment may be administeredconcurrently with the second therapeutic agent. In another specificembodiment, the second therapeutic agent is administered prior orsubsequent to administration of the anti-CD40 antibody or its fragment.

The antibodies and antibody fragments described herein can be formulatedor administered in combination with an immunosuppressant. Examples ofimmunosuppressants include, but are not limited to, calcineurininhibitors (e.g., cyclosporin A (Sandimmune®), cyclosporine G tacrolimus(Prograf®, Protopic®)), mTor inhibitors (e.g., sirolimus (Rapamune®,Neoral®), temsirolimus (Torisel®), zotarolimus, and everolimus(Certican®)), fingolimod (Gilenya™), myriocin, alemtuzumab (Campath®,MabCampath®, Campath-1H®), rituximab (Rituxan®, MabThera®), an anti-CD4monoclonal antibody (e.g., HuMax-CD4) an anti-LFA1 monoclonal antibody(e.g., CD11a), an anti LFA3 monoclonal antibody, an anti-CD45 antibody(e.g., an anti-CD45RB antibody), an anti-CD19 antibody (see, e.g., U.S.Patent Publication 2006/0280738), monabatacept (Orencia®), belatacept,indolyl-ASC (32-indole ether derivatives of tacrolimus and ascomycin),azathioprine (Azasan®, Imuran®), lymphocyte immune globulin andanti-thymocyte globulin [equine] (Atgam®), mycophenolate mofetil(Cellcept®), mycophenolate sodium (Myfortic®), daclizumab (Zenapax®),basiliximab (Simutlec®), cyclophosphamide (Endoxan®, Cytoxan®, Neosar™,Procytox™, Revimmune™), prednisone, prednisolone, leflunomide (Arava®),FK778, FK779, 15-deoxyspergualin (DSG), busulfan (Myleran®, Busulfex®),fludarabine (Fludara®), methotrexate (Rheumatrex®, Trexall®), etanercept(Enbrel®), adalimumab (Humira®), 6-mercaptopurine (Purinethol®),15-deoxyspergualin (Gusperimus), LF15-0195, bredinin, brequinar, andmuromonab-CD3 (Orthoclone®).

Methods for assessing immunosuppressive activity of an agent are knownin the art. For example, the length of the survival time of thetransplanted organ in vivo with and without pharmacological interventionserves as a quantitative measure for the suppression of the immuneresponse. In vitro assays may also be used, for example, a mixedlymphocyte reaction (MLR) assay (see, e.g., Fathman et al., J. Immunol.118:1232-8, 1977); a CD3 assay (specific activation of immune cells viaan anti-CD3 antibody (e.g., OKT3)) (see, e.g., Khanna et al.,Transplantation 67:882-9, 1999; Khanna et al. (1999) Transplantation67:S58); and an IL-2R assay (specific activation of immune cells withthe exogenuously added cytokine IL-2) (see e.g., Farrar et al., JImmunol 126:1120-5, 1981).

Cyclosporine A (CsA; CAS No. 59865-13-3, U.S. Pat. No. 3,737,433) andits analogs may be used as an immunosuppressant. A number of othercyclosporines and their derivatives and analogs that exhibitimmunosuppressive activity are known. Cyclosporines and theirformulations are described, for example, in 2004 Physicians' DeskReference® (2003) Thomson Healthcare, 58th ed., and U.S. Pat. Nos.5,766,629; 5,827,822; 4,220,641; 4,639,434; 4,289,851; 4,384,996;5,047,396; 4,388,307; 4,970,076; 4,990,337; 4,822,618; 4,576,284;5,120,710; and 4,894,235.

Tacrolimus (FK506) is a macrolide which exerts effects largely similarto CsA, both with regard to its molecular mode of action and itsclinical efficacy (Liu, Immunol. Today 14:290-5, 1993; Schreiber et al.,Immunol. Today, 13:136-42, 1992); however, these effects are exhibitedat doses that are 20 to 100 times lower than CsA (Peters et al., Drugs46:746-94, 1993). Tacrolimus and its formulations are described, forexample, in 2004 Physicians' Desk Reference® (2003) Thomson Healthcare58th ed, and U.S. Pat. Nos. 4,894,360; 4,929,611 and 5,164,495.

Sirolimus (rapamycin) is an immunosuppressive lactam macrolideproduceable, for example by Streptomyces hygroscopicus. Numerousderivatives of sitolimus and its analogs and their formulations areknown and described, for example, in 2004 Physicians' Desk Reference®(2003) Thomson Healthcare, 58th ed., European Patent EP 0467606, PCTPublication Nos. WO 94/02136, WO 94/09010, WO 92/05170, WO 93/11130, WO94/02385, WO 95/14023 and WO 94/02136, and U.S. Pat. Nos. 5,023,262;5,120,725; 5,120,727, 5,177,203; 5,258,389; 5,118,677; 5,118,678;5,100,883; 5,151,413; 5,120,842; and 5,256,790.

In some embodiments, the second agent is a cytotoxic agent which may bea convention chemotherapeutic such as, for example, doxorubicinpaclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C oretoposide. In addition, potent agents such as CC-1065 analogues,calicheamicin, maytansine, analogues of dolastatin 10, rhizoxin, andpalytoxin can be linked to the anti-CD40 antibodies or agents thereof.

In additional embodiments, the second agent is a humanized anti-HER2monoclonal antibody, RITUXAN (rituximab: Genentech, Inc., South SanFrancisco, Calif.), a chimeric anti-CD20 monoclonal antibody), OVAREX(AltaRex Corporation, MA); PANOREX (Glaxo Wellcome, N.C.; a murine IgG2aantibody); ERBITUX (cetuximab) (Imclone Systems Inc., N.Y.; an anti-EGFRIgG chimeric antibody); VITAXIN (MedImmune, Inc., MD); CAMPATH I/H(Leukosite, Mass.; a humanized IgG1 antibody); Smart MI95 (ProteinDesign Labs, Inc., CA; a humanized anti-CD33 IgG antibody); LymphoCide(Immunomedics, Inc., NJ; a humanized anti-CD22 IgG antibody); Smart ID10(Protein Design Labs, inc., CA; a humanized anti-HLA-DR antibody),Oncolym (Techniclone, Inc., CA; a radiolabeled murine anti-HLA-Dr10antibody); ALLOMUNE (BioTransplant, Calif.; a humanized anti-CD2 mAb):AVASTIN (Genemech, Inc., CA; an anti-VBGF humanized antibody);Epratuzamab (Imnunomedics, Inc., NJ and Amgen, CA; an anti-CD22antibody); and CEAcide (Immunonedics, NJ; a humanized anti-CEAantibody).

Other suitable antibodies that may be used as the second agent include,but are not limited to, antibodies against the following antigens:CA125, CA15-3, CA19-9, L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242placental alkaline phosphatase, prostate specific antigen prostatic acidphosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4,anti-transferrin receptor, p97, MUC1-KLH, CEA, gp100, MART1, ProstateSpecific Antigen IL-2 receptor, CD20, CD52, CD33, CD22, human chorionicgonadotropin, CD38, mucin, P21, MPG, and Neu oncogene product.

Non-Therapeutic Uses

The antibodies described herein are useful as affinity purificationagents. In this process, the antibodies or fragments thereof areimmobilized on a solid phase such a Protein A resin, using methods wellknown in the art. The immobilized antibody or its fragment is contactedwith a sample containing the CD40 protein (or fragment thereof) to bepurified, and thereafter the support is washed with a suitable solventthat will remove substantially all the material in the sample except theCD40 protein, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release theCD40 protein from the antibody.

The present anti-CD40 antibodies are also useful in diagnostic assays todetect and/or quantify CD40 protein, for example, detecting CD40expression in specific cells, tissues, or serum.

The antibodies described herein may be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays. See. e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Pharmaceutical Compositions

The present disclosure provides a composition, e.g., a pharmaceuticalcomposition, containing an antibody, or antigen-binding portion(s)thereof, of The present disclosure, formulated together with apharmaceutically acceptable carrier. In another embodiment, thecomposition may contain an isolated nucleic acid encoding the presentantibody or antigen-binding portion thereof, and a pharmaceuticallyacceptable carrier. The composition may be effective to reduce thelikelihood of or increase the duration prior to transplant rejection, toinduce immunosuppression, or to treat an autoimmune disorder in asubject. The present composition may be effective in any of the methodsdescribed herein.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that, arephysiologically compatible. Depending on the route of administration thepresent antibodies (or antigen-binding portion(s) thereof) may be coatedin a material to protect the antibodies (or antigen-binding portion(s)thereof) from the action of acids and other natural conditions that mayinactivate the antibodies (or antigen-binding portion(s) thereof). Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.In certain embodiments, the present composition may include isotonicagents, for example, sugars, polyaleohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin.

Pharmaceutical compositions of the invention may contain the presentantibody or its fragment, and the second therapeutic agent as describedherein (e.g., one or more immunosuppressants).

The composition may be in the form of a solution, a suspension, anemulsion, an infusion device, or a delivery device for implantation, orit may be presented as a solid form (e.g., a dry powder) to bereconstituted with water or another suitable vehicle before use. Thecompositions may be in the form of an oil emulsion, water-in-oilemulsion, water-in-oil-in-water emulsion, site-specific emulsion,long-residence emulsion, stickyemulsion, microemulsion, nanoemulsion,liposome, microparticle, microsphere, nanosphere, nanoparticle andvarious natural or synthetic polymers, such as nontesorbable impermeablepolymers such as ethylenevinyl acetate copolymers and Hytrel®copolymers, swellable polymers such as hydrogels, or resorbable polymerssuch as collagen and certain polyacids or polyesters such as those usedto make resorbable sutures, that allow for sustained release of thevaccine.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration or a liquid forintravenous intrathecal, subcutaneous or parenteral administration, or apolymer or other sustained release vehicle for local administration.

In one aspect, a solution of the composition is dissolved in apharmaceutically acceptable carrier, e.g., an aqueous carrier if thecomposition is water-soluble. Examples of aqueous solutions include,e.g., water saline, phosphate buffered saline, Hank's solution, Ringer'ssolution, dextrose/saline, glucose solutions and the like. Theformulations can contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such asbuffering agents, tonicity adjusting agents, wetting agents, detergentsand the like. Additives can also include additional active ingredientssuch as bactericidal agents, or stabilizers. For example, the solutioncan contain sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate or triethanolammeoleate. Solid formulations can be used in The present disclosure. Theycan be formulated as, e.g., pills, tablets, powders or capsules. Forsolid compositions, conventional solid carriers can be used whichinclude, e.g., mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, andthe like. Suitable pharmaceutical excipients include e.g., starch,cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, magnesium stearate, sodium stearate glycerolmonostearate, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.).

In one aspect, the pharmaceutical formulations comprising compositionsor nucleic acids polypeptides, or antibodies of the invention areincorporated in lipid monolayers or bilayers. e.g., liposomes. U.S. Pat.Nos. 6,110,490; 6,096,716; 5,283,185 and 5,279,833. Aspects of theinvention also provide formulations in which water soluble nucletcacids, peptides or polypeptides of the invention have been attached tothe surface of the monolayer or bilayer. For example, peptides can beattached to hydrazide-PEG-(distearoylphosphatidyl)ethanolamine-containing liposomes (see, e.g., Zalipsky, Bioconjug. Chem6: 705-708, 1995). Liposomes or any form of lipid membrane, such asplanar lipid membranes or the cell membrane of an intact cell, e.g., ared blood cell, can be used. Liposomal formulations can be by any means,including admimstration intravenously, transdermally (see e.g., Vutla,J. Pharm. Sci. 85: 5-8, 1906), transmucosally, or orally. The inventionalso provides pharmaceutical preparations in which the nucleic acid,peptides and/or polypeptides of the invention are incorporated withinmicelles and or liposomes (see, e.g. Suntres, J. Pharm. Pharmacol 46:23-28, 1904, Woodle. Pharm. Res. 9; 260-265, 1992). Liposomes andliposomal formulations can be prepared according to standard methods andare also well known in the art. Akimaru, Cytokines Mol. Ther. 1;197-210, 1995. Alving, Immunol. Rev. 145: 5-31, 1995. Szoka, Ann. Rev.Biophys. Bioeng. 9: 467, 1980. U.S. Pat. Nos. 4,235,871; 4,501,728 and4,837,028.

In one aspect, the compositions are prepared with carriers that willprotect the peptide against rapid elimination from the body, such as acontrolled release formulation including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. U.S. Pat. No. 4,522,811.

A composition of The present disclosure can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. Administration may be parenteral, intravenous,intrathecal, subcutaneous, oral, topical, local, intramuscular,intradermal, transdermal, subdermal, rectal spinal, or epidermal.Intravenous delivery by continuous infusion is one exemplary method foradministering the present antibodies.

To administer the present agent by certain routes of administration, itmay be necessary to coat the agent with, or co-administer the agentwith, a material to prevent its inactivation. For example, the agent maybe administered to a subject in an appropriate carrier, for example,liposomes, or a diluent. Pharmaceutically acceptable diluents includesaline and aqueous buffet solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., J. Neuroimmunol. 7:27-41, 1984)

Parenteral administration can include modes of administration other thanenteral and topical administration, usually by injection, and include,without limitation, intravenous, intramuscular intraarterialintrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrastemal injection and infusion. Examples of suitable aqueous andnonaqueous earners which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

Methods for preparing parenterally administrable compositions will beknown or apparent to those skilled in the art and are described indetail. Bai, J. Neuroimmunol. 80: 65-75, 1997. Warren. J Neurol. Sci.152: 31-38, 1997. Tonegawa, J. Exp. Med. 186:507-515, 1997. Formulationsfor parenteral administration may, for example, contain excipients,sterile water, saline polyalkylene glycols such as polyethylene glycol,oils of vegetable origin, or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the present agent. Nanoparticulate formulations (e.g.,biodegradable nanoparticles, solid lipid nanoparticles, liposomes) maybe used to control the biodistribution of the present agent. Otherpotentially useful delivery systems include ethylene-vinyl acetatecopolymer particles, osmotic pumps, intrathecal pumps, implantableinfusion systems, and liposomes. The concentration of the agent in theformulation varies depending upon a number of factors, including thedosage of the drug to be administered, and the route of administration.

Sterile injectable solutions can be prepared by incorporating thepresent agent in the required amount in an appropriate solvent with oneor a combination of ingredients enumerated above, as required, followedby sterilization microfiltration. Generally, dispersions are prepared byincorporating the present agent into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation include vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. Dosage regimens areadjusted to provide the optimum desired response (e.g., a therapeuticresponse). For example, a single bolus may be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. For example, the antibodies of the inventionmay be administered once or twice weekly by subcutaneous injection oronce or twice monthly by subcutaneous injection. Parenteral compositionsmay be formulated in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of active agentcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical earner. The specification for the dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active agent and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of agenting such an active agent for the treatment ofsensitivity in individuals.

When administered orally, the present compositions may be protected fromdigestion. This can be accomplished either by complexing the antibody orantigen-binding portion thereof with a composition to render itresistant to acidic and enzymatic hydrolysis or by packaging theantibody or antigen-binding portion thereof in an appropriatelyresistant carrier such as a liposome. Means of protecting agents fromdigestion are well known in the art. Fix, Pharm Res. 13:1760-1764, 1996.Samanen, J. Pharm. Pharmacol. 48:119-135, 1996, U.S. Pat. No. 5,391,377.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated can be used in the formulation. Suchpenetrants are generally known in the art, and include, e.g., fortransmucosal administration, bile salts and fusidic acid derivatives. Inaddition, detergents can be used to facilitate permeation. Transmucosaladministration can be through nasal sprays or using suppositories.Sayani Crit. Rev. Ther. Drug Carrier Syst. 13: 85-184, 1996. Fortopical, transdermal administration, the agents are formulated intoointments, creams, salves, powders and gels. Transdermal deliverysystems can also include, e.g., patches.

The present compositions can also be administered in sustained deliveryor sustained release mechanisms. For example, biodegradeablemicrospheres or capsules or other biodegradeable polymer configurationscapable of sustained delivery of a peptide can be included in theformulations of the invention (see. e.g., Putney, Nat. Biotechnol. 16:153-157, 1998).

For inhalation, the present compositions can be delivered using anysystem known in the art, including dry powder aerosols, liquids deliverysystems, air jet nebulizers, propellant systems, and the like. Patton,Biotechniques 16:141-143, 1998. Also can be used in The presentdisclosure are product and inhalation delivery systems for polypeptidemacro molecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.),Aradigrn (Hayward, Calif.), Aerogen (Santa Clara, Calif.), InhaleTherapeutic Systems (San Carlos, Calif.), and the like. For example, thepharmaceutical formulation can be administered in the form of an aerosolor mist. For aerosol administration, the formulation can be supplied infinely divided form along with a surfactant and propellant. In anotheraspect, the device for delivering the formulation to respiratory tissueis an inhaler in which the formulation vaporizes. Other liquid deliverysystems include, e.g., air jet nebulizers.

Compositions can be administered in a single dose treatment or inmultiple dose treatments on a schedule and over a time periodappropriate to the age, weight and condition of the subject, theparticular composition used, and the route of administration. Thefrequency of administration can vary depending on any of a variety offactors, e.g., severity of the symptoms, degree of immunoprotectiondesired, whether the composition is used for prophylactic or curativepurposes, etc. For example, in one embodiment, the composition accordingto the invention is administered once per month, twice per month, threetimes per month, every other week (qow) once per week (qw), twice perweek (biw), three times per week (tiw), four times per week, five timesper week, six times per week, every other day (qod), daily (qd), twice aday (qid), or three times a day (tid).

The duration of administration of a polypeptide according to theinvention, e.g., the period of time over which the composition isadministered, can vary, depending on any of a variety of factors, e.g.,subject response, etc. For example, the composition can be administeredover a period of time ranging from about one day to about one week, fromabout two weeks to about four weeks, from about one month to about twomonths, from about two months to about four months, from about fourmonths to about six mouths, from about six months to about eight months,from about eight months to about 1 year, from about 1 year to about 2years, or from about 2 years to about 4 years or more.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular agent being employed, the duration of the treatment, otherdrugs, agents and/or materials used its combination with the particularcompositions employed, the age, sex, weight, condition, general healthand prior medical history of the patient being treated, and like factorswell known in the medical arts. A physician or veterinarian havingordinary skill in the art can readily determine and prescribe theeffective amount of the pharmaceutical composition required. Forexample, the physician or veterinarian can start doses of the agents ofthe invention employed in the pharmaceutical composition at levels lowerthan that required in order to achieve the desired therapeutic effectand gradually increase the dosage until the desired effect is achieved.In general, a suitable daily dose of a composition of the invention willbe that amount of the agent which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. If desired, the effective dailydose of a therapeutic composition may be administered as two, three,four, five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. In oneembodiment, the dosage of such agents lies within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. In another embodimentthe therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test agent which achieves a half-maximal inhibitionof symptoms) as determined in cell culture. Sonderstrup, Springer, Sem,Immunopathol. 25: 35-45, 2003. Nikula et al., Inhal. Toxicol 4(12):123-53, 2000.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antigen-bindingportion of the invention is from about 0.001 to about 100 mg/kg bodyweight or more, about 0.1 to about 100 mg/kg body weight, about 0.01 toabout 80 mg/kg body weight, about 0.001 to about 60 mg/kg body weight,about 0.01 to about 30 mg/kg body weight, about 0.01 to about 25 mg/kgbody weight, about 0.5 to about 25 mg/kg body weight about 0.1 to about15 mg/kg body weight, about 0.1 to about 20 mg/kg body weight, about 10to about 20 mg/kg body weight, about 0.75 to about 10 mg/kg body weight,about 1 to about 10 mg kg body weight, about 2 to about 9 mg/kg bodyweight, about 1 to about 2 mg/kg body weight, about 3 to about 8 mg/kgbody weight, about 4 to about 7 mg/kg body weight, about 5 to about 6mg/kg body weight, about 8 to about 13 mg/kg body weight, about 8.3 toabout 12.5 mg/kg body weight, about 4 to about 6 mg/kg body weight,about 4.2 to about 6.3 mg/kg body weight, about 1.6 to about 2.5 mg/kgbody weight, about 2 to about 3 mg/kg body weight, or about 10 mg/kgbody weight. The dosage administered to a subject may also be about 0.1mg/kg to about 50 mg/kg, about 1 mg/kg to about 30 mg/kg about 1 mg/kgto about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, or about 1 mg/kg toabout 10 mg/kg of the subject's body weight. Exemplary doses include,but are not limited to, from 1 ng/kg to 100 mg/kg. In some embodiments,a dose is about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg,about 4 mg/kg about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg,about 13 mg/kg, about 14 mg/kg, about 15 mg/kg or about 16 mg/kg of thesubject's body weight WO 94/04188.

The composition is formulated to contain an effective amount of thepresent antibody or antigen-binding portion thereof, wherein the amountdepends on the animal to be treated and the condition to be treated. Inone embodiment, the present antibody or antigen-binding portion thereofis administered at a dose ranging hum about 0.01 mg to about 10 g, fromabout 0.1 mg to about 9 g, from about 1 mg to about 8 g, from about 1 mgto about 7 g, from about 5 mg to about 6 g, from about 10 mg to about 5g, from about 20 mg to about 1 g, from about 50 mg to about 800 mg, fromabout 10 mg to about 500 mg, from about 0.01 mg to about 10 g, fromabout 0.05 μg to about 1.5 mg, from about 10 μg to about 1 mg protein,from about 30 μg to about 500 μg, from about 40 pg to about 300 pg, fromabout 0.1 μg to about 200 mg, from about 0.1 μg to about 5 μg, fromabout 5 μg to about 10 μg, from about 10 μg to about 25 μg, from about25 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100μg to about 500 μg, from about 500 μg to about 1 mg from about 1 mg toabout 2 mg. The specific dose level for any particular subject dependsupon a variety of factors including the activity of the specificpeptide, the age, body weight, general health, sex, diet, time ofadministration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

Articles of Manufacture

In another aspect, an article of manufacture containing materials usefulfor the treatment of the conditions or disorders described herein isincluded. The article of manufacture comprises a container and a label.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition that iseffective for treating the condition and may have a sterile access port.For example, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle. The activeagent in the composition may be the humanized anti-CD40 antibody or itsfragment, or any other antibody or its fragment as described herein. Thelabel on or associated with the container indicates that the compositionis used for treating the condition of choice. The article of manufacturemay further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

In one embodiment, the invention provides for a kit containing ananti-CD40 antibody or antigen-binding portion thereof. Additionalcomponents of the kits may include one or more of the followinginstructions for use; other reagents, a therapeutic agent, or an agentuseful for coupling an antibody to a label or therapeutic agent, orother materials for preparing the antibody for administration;pharmaceutically acceptable carriers; and devices or other materials foradministration to a subject.

The kit may or may not contain the second therapeutic agent as describedherein. The agents can be mixed together, or packaged separately withinthe kit.

The kit may or may not contain at least one nucleic acid encodinganti-CD40 antibodies or fragment thereof, and instructions forexpression of the nucleic acids. Other possible components of the kitinclude expression vectors and cells.

The present antibody or its fragment can be used in a diagnostic kit,i.e., a packaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit may include substrates and cofactorsrequired by the enzyme such as a substrate precursor that provides thedetectable chromophore or fluorophore. In addition, other additives maybe included such as stabilizers, buffers (for example a block buffer orlysis buffer), and the like. The relative amounts of the variousreagents may be varied widely to provide for concentrations in solutionof the reagents that substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders, usually lyophilized,including excipients that on dissolution will provide a reagent solutionhaving the appropriate concentration.

Epitope Mapping

Methods for identifying the particular epitope to which an antibodybinds are known to those skilled in the art. Standard techniques includepeptide scanning, in which overlapping, short peptides (for example,10-30 amino acids, e.g., 20, in length) derived from the full lengthprotein to which the antibody binds are individually tested for theirability to bind the antibody. From such experiments, the region of theprotein to which the antibody binds can then be determined.

Site-directed mutagenesis can also be used to identify the antigenicregion(s) of a particular protein. In this approach, point mutations aresystematically introduced into the target polypeptide and the ability ofthe antibody to bind the peptide with mutations at various positions isused to determine whether a particular region of that protein containsthe epitope to which the antibody binds.

Antibody epitopes can also be identified using high-throughputmutagenesis techniques, such as Shotgun Mutagenesis (Integral Molecular,Inc., Philadelphia, Pa.), which can be used to generate huge numbers ofmutations within the target protein. Such methodologies permit efficientidentification of epitopes within the protein.

To determine if various antibodies to CD40 bind similar epitopes, an invitro competitive blockade assay may be performed. In one embodiment,the antibodies 2C10, 3A8 and Chi220, a chimeric IgG1 CD40-specificantibody, were used in the assay. 2C10 was conjugated to allophycocyanin(APC) using the Lightning Link antibody labeling kit (Novus Biologics,Littleton, Co.). Human PBMCs were incubated with escalatingconcentrations of 2C10, 3A8, or Chi220, and then stained with theAPT-conjugated 2C10 to assess the ability of each antibody tocross-block 2C10. Binding of APC-conjugated 2C10 decreased withincreasing concentrations of 2C10 but not Chi220 or 3A8 as shown in FIG.12. The result indicates that 2C10 binds a unique epitope distinct fromeither Chi220 or 3A8.

CD40 Fragments

The invention also features fragments of CD40 that include the epitopethat is specifically bound by the 2C10 antibody. The 2C10 antibody wasraised against the extracellular portion of the CD40 polypeptide. The2C10 antibody reacts with a portion of this sequence (SEQ ID NOs: 5 and6).

The disclosure therefore features CD40 fragments (e.g., fewer than 150,120, 100, 80, 70, 60, 50, 40, 30, 20, 18, 15, 12, 11, 10, 9, 8, or 7)amino acids in length that are specifically bound by the 2C10 antibody.In certain embodiments, the fragment is 8-10, 8-12, 8-15, 8-20, 8-30,8-40, 8-50, 8-60, 8-70, 8-80, or 8-100 amino acids in length. In otherembodiments, the fragment is 7-10, 7-12, 7-15, 7-20, 7-30, 7-40, 7-50,7-60, 7-70, 7-80 or 7-100 in length. The 2C10 antibody binds to anepitope present within the sequence of amino acids 8-10, 8-12, 8-15,8-20, 8-30, 8-40, 8-50, 8-60, 8-70, 8-80, 8-100, 7-10, 7-12, 7-15, 7-20,7-30, 7-40, 7-50, 7-60, 7-70, 7-80, or 7-100, of SEQ ID NO: 6.

The invention also features fusion protein that includes a fragmentdescribed herein and a heterologous sequence. In certain embodiments,one of the fusion partners is the Fc protein (e.g., mouse Fc or humanFc). The fusion may also be a sequence useful for antibody production,e.g., a maltose binding protein or GST. In other embodiments, the fusionprotein is a purification or detection tag, for example, proteins thatmay be detected directly or indirectly such as green fluorescentprotein, hemagglutinin, or alkaline phosphatase), DNA binding domains(for example, GAL4 or LexA), gene activation domains (for example, GAL4or VP16), purification tags, or secretion signal peptides (e.g.,preprotyrypsin signal sequence). In other embodiments, the fusionpartner may be a tag, such as c-myc, poly histidine, or FLAG. Eachfusion partner may contain one or more domains e.g., a preprotrypsinsignal sequence and FLAG tag.

The CD40 fragments and fusion proteins described herein may be producedby transformation of a suitable host cell with a polynucleotide moleculeencoding the polypeptide fragment or fusion protein in a suitableexpression vehicle.

Any of a wide variety of expression systems may be used. Exemplaryexpression systems include prokaryotic hosts (e.g., E. coli) andeukaryotic hosts (e.g., S. cervisiae, insect cells, e.g., Sf21 cells, ormammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Suchcells are available from a wide range of sources (e.g., the AmericanType Culture Collection, Manassas, Va.). The method of transformation ortransfection and the choice of expression vehicle will depend on thehost system selected. Transformation and transfection methods aredescribed e.g., in Kucherlapti et al. (CRC Crit. Rev. Biochem.16:349-379, 1982) and in DNA Transfer to Cultured Cells (eds., Ravid andFreshney, Wiley-Liss, 1998); and expression vehicles may be chosen fromthose provided, e.g., in Vectors: Expression Systems: EssentialTechniques (ed., Jones, Wiley & Sons Ltd., 1998).

Once the recombinant CD40 polypeptide fragment or fusion protein isexpressed, it can be isolated e.g., using affinity chromatography. Inone example, an antibody specific to CD40 (e.g., an antibody or itsfragment as described herein) may be attached to a column and used toisolate the polypeptide fragment or fusion protein. Lysis andfractionation of fragment- or fusion protein-harboring cells prior toaffinity chromatography may be performed by standard methods (see e.g.,Methods in Enzymeology, volume 182, eds., Abelson, Simon, and Deutscher,Elsevier, 1990). Once isolated, the CD40 polypeptide fragment or fusionprotein can, if desired, be further purified, e.g., by high performanceliquid chromatography (see e.g., Fisher, Laboratory Techniques inBiochemistry and Molecular Biology, eds., Work and Burdon, Elsevier,1980; and Scopes, Protein Purification: Principles and Practice, ThirdEdition, ed., Cantor, Springer, 1994).

The CD40 polypeptide fragments or fusion proteins can also be producedby chemical synthesis (e.g., by the methods described in Solid PhasePeptide Synthesis, 2nd ed., 1984. The Pierce Chemical Co., Rockford,Ill.; and Solid-Phase Synthesis: A Practical Guide, ed., Kates andAlbericio, Marcel Dekker Inc., 2000).

The present antibodies, antigen-binding portions thereof, compositionsand methods can be used in all vertebrates, e.g., mammals andnon-mammals including human, mice, rats, guinea pigs, hamsters, dogscats, cows, horses, goats, sheep, pigs, monkeys, apes, gorillas,chimpanzees, rabbits, ducks, geese, chickens, amphibians, reptiles andother animals.

The following examples of specific aspects for carrying out the presentdisclosure are offered for illustrative purposes only, and are notintended to limit the scope of the present disclosure in any way.

Example 1 Production and Identification of Anti-CD40 Murine Antibodies

Mice (strain AJ) were immunized with a fusion protein consisting of theextracellular domain of rhesus macaque (M. mulatia) CD40 (amino acidsequence:

EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCSESEFLDTWNRETRCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGLHCMSESCESCV: SEQ ID NO: 5) fused to maltosebinding protein (CD40-MBP). The amino acid sequence in this region ofthe rhesus macaque CD40 protein differs from human CD40 protein at fiveamino acid positions (human amino acid sequence:EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETD TICTCEEGWHCTSEACESCV; SEQ ID NO: 6). CD40-MBP wasadministered to mice multiple times with complete Freund's adjuvant andincomplete Freund's adjuvant Splenocytes from immunized mice were fusedwith the mouse myeloma cell line SP2/0 and hybrids selected usingstandard hybridoma technology.

Antibodies were selected for reactivity in a second fusion proteinconsisting of the same rhesus CD40 domain fused to glutamine synthetase(CD40-GST). Antibodies reactive to CD40-GST by ELISA were further testedfor reactivity to native CD40 expressed on rhesus macaque blood B cells,human blood B cells and rhesus macaque B-lymphoblastoid cell lines byflow cytometry. As a final level of selection, antibodies were tested inan in vitro assay for their ability to inhibit human or rhesus macaque Bcell activation after co-culture CD154-expressing Jurkat D1.1 cells. Astable subclone of anti-CD40 antibody 2C10 was obtained by limitingdilution. The antibody is a mouse IgG1-kappa.

Lowe et. al., A novel monoclonal antibody to CD40 prolongs isletallograft survival. Am. J Transplant (2012) 12(8):2079-87

Antibody Cloning

Variable regions of monoclonal antibodies can be cloned using any methodknown in the art. PCR-based methods for obtaining antibody variableregion sequences for hybridoma cells are described, for example, inLarrick et al., Nat. Biotechnol. 7:934-8, 1989 and in Orlandi et al.,Proc. Natl. Acad. Sci. USA 86:3833-7, 1989. Using these techniques orsimilar techniques, the variable regions of monoclonal antibodies can becloned and subject to further manipulation. In the present case, thevariable sequences from the heavy and light chains of the 2C10 antibodywere cloned and were sequenced. The DNA representing the immunoglobulinheavy and light chain variable regions from the 2C10 hybridoma werecloned using 5′ RACE PCR employing the following DNA primers:

Mouse kappa reverse: (SEQ ID NO: 7)5′-CTA ACA CTC ATT CCT GTT GAA GCT CTTGAC; Mouse kappa forward:(SEQ ID NO: 8) 5′-GCT GAT GCT GCA CCA ACT GTA TCC-3′ Mouse IgG1 reverse:(SEQ ID NO: 9) 5′-GGC AAC GTT GCA GGT CTC GC-3′ Mouse IgG1 forward:(SEQ ID NO: 10) 5′-CTG GAT CTG CTG CCC AAA CTA ACT CC-3′

PCR products were cloned into a commercial cloning vector and weresequenced using standard sequencing techniques. The resulting sequencesare provided in FIG. 1.

The immunoglobulin variable region genes were cloned from the hybridomassecreting anti-CD40 antibody clone 2C10 and from anti-human CD40 clone3A8 (Kwekkeboom et al., Immunology 79:439-44, 1993) (obtained from theAmerican Type Culture Collection, ATCC, Vienna, Va.) using 5′ rapidamplification of cDNA ends-polymerase chain reaction. The immunoglobulinheavy and light chain variable regions were subcloned into expressionvectors containing rhesus IgG1 or rhesus IgG4 heavy chain and rhesuskappa light chain constant region sequences.

Recombinant heavy and light chains were subcloned into expressionvectors and packaged in retroviral vectors used to transduce Chinesehamster ovary cells using the GPEx™ expression technology (CatalentPharma Solutions, Middleton, Wis.). A pool of transduced cells was grownin serum-free medium and secreted antibody was purified by protein Aaffinity chromatography. The purified chimeric rhesus IgG1 (2C10R1,3A8R1) and IgG4 (2C10R4) antibodies were diafiltered into phosphatebuffer; endotoxin levels were confirmed to be less than 1 endotoxinunit/mg.

Antibody Characterization

2C10 Binds to CD40 and Prevents Binding of CD154

To assess the ability of 2C10 to bind to both rhesus and human CD40,recombinantly expressed human or rhesus CD40 were adsorbed to ELISAplates and reacted with varying concentrations of 2C10. Binding of 2C10to CD40 was detected using goat anti-mouse IgG-HRP in an ELISA. Theresults in FIG. 2B show that 2C10 has similar binding affinities torhesus and human CD40, which is important for clinical translation of2C10. To confirm the ability of 2C10 to block binding of its cognateligand, CD154, rhesus and human B cells were incubated with escalatingconcentrations of 2C10 or an isotype control and then incubated withhistidine-tagged soluble CD154 (R&D Systems, Minneapolis, Minn.) andanalyzed for histidine expression. 2C10 blocked the binding of CD154 ina dose-dependent manner (FIG. 3), indicating that 2C10 can effectivelyblock the interaction of T cell-bound CD154 with CD40 on B cells andantigen-presenting cells.

TABLE 2 CD40 Receptor Binding Kinetics of 2C10 Anti-CD40 Antibodies 2C103A8 5D12 4D11 Chi220 K_(on) (M⁻¹ s⁻¹) 2.73 × 0.223 × 1.51 × 1.25 × 0.413× 10³ 10³ 10³ 10³ 10³ K_(off) (s⁻¹) 1.86 × 4.15 × 1.54 × 1.58 × 2.5 ×10⁻⁶ 10⁻⁶ 10⁻⁵ 10⁻⁶ 10⁻⁵ K_(D) (M) 2.73 × 1.86 × 1.02 × 2.28 × 6.07 ×10⁻¹⁰ 10⁻⁸ 10⁻⁸ 10⁻⁹ 10⁻⁸2C10 Blocks B Cell Activation in Rhesus Monkey and Human PeripheralBlood Mononuclear Cells

The anti-CD40 antibody 2C10 was characterized with respect to itsability to affect B cell activation both using rhesus monkey and humanperipheral blood mononuclear cells (PBMCs). CD20 expression was chosenas being an indicator of B cells, and expression of CD23, CD80, and CD86is associated with B cell activation. 2C10 was first assessed for itsability to bind to CD20. Rhesus or human PBMCs were incubated withfluorochrome-conjugated 2C10 and an anti-CD20 antibody. Flow cytometricanalysis was used to confirm the binding of 2C10 to human and rhesusCD20+ B cells (FIG. 2A). In another set of experiments, PBMCs fromeither rhesus monkey or humans were cultured either in the presence orabsence of CD154⁺ Jurkat D1.1 cells, an immortalized T lymophocyte cellline. Activation of B cells was determined by measuring expression ofthree markers (CD23, CD80, and CD86) in CD20+ cells present in thePBMCs. The general scheme of this assay is shown in FIG. 4. As shown inFIG. 4, culturing PBMCs in the presence of Jurkat cells resulted inincreased expression of all three markets, indicating that B cells areactivated by the CD154 Jurkat cells.

To test the ability of antibodies to block B cell activation, PBMCs andJurkat cells were co-cultured in the presence or absence of one of threeantibodies: 3A8, 5C8, and 2C10. The 3A8 antibody is a mouse anti-humanCD40 antibody (ATCC Deposit No. HB-12024), and 5C8 is an anti-CD154antibody (ATCC Deposit No. CRL-10915). Each was used as a positivecontrol. Co-cultures were conducted over a range of five orders ofmagnitude of antibody concentration (0.001 μg to 10 μg). As shown inFIG. 5, 3A8 did not block B cell activation in rhesus PBMCs, as measuredby CD23 expression, whereas both 2C10 and 5C8 were able to blockactivation with similar efficiency. Corresponding changes were alsoobserved with CD80 and CD86 expression. These results indicate that 2C10binds to a different epitope on CD40 than 3A8. These results alsoindicate that 2C10 acts primarily as a CD40 antagonist in contrast to3A8 which has previously been shown to act as partial agonists with weakstimulatory potential (Adams et al., J. Immunol. 174:542-50, 2005,Badell et al., Am. J. Transplant accepted for publication, 2011). When asimilar experiment was performed using human, rather than rhesus, PBMCs,both 2C10 and 5C8 were again observed to block B cell activation, asmeasured by CD86 expression, with similar efficiency. Here, the 3A8antibody, unlike with the rhesus PBMCs, blocked B cell activation (FIG.6).

The 2C10 and 3A8 antibodies were also tested for their ability toactivate B cells in the absence of Jurkat cells using either rhesusmonkey or human PBMCs. Here, PBMCs were cultured either in the presenceor absence of either 2C10 or 3A8. Expression of CD23, CD80, and CD86 wasthen measured in CD20⁺ cells. As shown in FIG. 7, CD23 expression inrhesus cells was increased in the presence of the 3A8, but not the 2C10,antibody. By contrast, neither 3A8 nor 2C10 activated human B cells. Thedifferences in activity observed between the 3A8 and 2C10 antibodyindicate that the 2C10 antibody binds to an epitope different from thatof the 3A8 antibody.

2C10 Prevents a T Cell-Dependent Antibody Response

Having established that 2C10 binds to a unique epitope on CD40 inhibitsB cell activation similarly to an anti-CD154 antibody, and lacksagonistic properties, we then characterized the effects of 2C10 in vivo.Recombinant mouse-rhesus chimeric forms of 2C10 were generated usingeither rhesus IgG1 (2C10R1) or IgG4 (2C10R4) heavy chain and rhesuskappa light chain constant legion sequences. A chimeric rhesus IgG1 formof 3A8 (3A8R1) was also generated for use as a control.

Rhesus macaques were immunized once on day zero with4-hydroxy-3-nitrophenylacetyl-conjugated keyhole limpet hemocyanin (KLH,10 mg IM) antigen (Biosearch Technologies, Novato, Calif.). Prior toimmunization and at one week, cohorts of three animals received anintravenous dose (50 mg/kg) of 2C10R1, 2C10R4, 3A8R1, or saline. Allanimals were observed for 70 days, and flow cytometry was performedweekly. Treatment with either recombinant 2C10 isotypes resulted inmodest change in peripheral B cell counts (FIG. 8) compared to thepreviously reported significant and prolonged depletion of peripheral Bcells occurring in animals receiving either 3A8R1 (Badell et al., Am. J.Transplant. 10:214, 2010) or Chi220 (Adams et al., J. Immunol.174:542-50, 2005).

T cell-dependent antibody responses to KLH-NP were tested by ELISA.Plates were coated with KLH (0.01 mg/ml. Sigma, St. Louis, Mo.) andblocked with Super Block (Thermo Scientific, Woodstock, Ga.). Pre- andpost-treatment plasma samples were serially diluted, plated for 1 hr.and washed with phosphate-buffered saline/0.05% Tween. Anti-KLHantibodies were detected by incubating for 1 hr with monoclonalanti-rhesus IgG-horseradish peroxidase (clone 1B3, NHP Reagent Resource,Boston, Mass.). Plates were then incubated with Peroxidase SubstrateSolution (KPL). Stop solution (KPL) was then added, and optical densitywas read on an ELISA plate reader at 450 nm. A sample was consideredpositive at a given dilution if the optical density reading of thepost-treatment plasma exceeded the optical density of the pre-treatmentplasma at the same dilution by 2-fold. Following KLH immunization,control animals developed high-titer KLH-specific IgG (FIG. 9). Animalsthat received 3A8R1 also developed anti-KLH responses, but titers wereapproximately 10-fold lower than controls despite significant depletionof B cells. In contrast, the generation of IgG anti-KLH antibodies wasnearly completely blocked through day 56 in all animals that receivedeither 2C10R1 or 2C10R4.

2C10 Significantly Prolongs Islet Allograft Survival in a Macaque Modelof Allogeneic Islet Transplantation

We further tested 2C10R4, the CD4 purified chimeric rhesis IgG4antibody, in a nonhuman primate allogenic islet transplant model (FIG.10). Rhesus macaques weighing 10-20 kg underwent donor pancreatectomyone day prior to non-plantation via a midline laparotomy. The pancreaswas isolated and placed on ice after the animals were terminallyexsanguinated. Islet isolation was performed using Collagenase Neutralprotease (950 Wunsch units and 63 units, respectively; Serva,Heidelberg, Germany). The digested pancreas was purified on a fourlayer, discontinuous Euroficoll gradient (Mediateh, Manassas, Va.) andCobe 2901 blood cell processor (CaridianBCT, Lakewood, Col.) Samples ofthe final islet preparation were counted and expressed as isletequivalents (IEQ). Isolated islets were cultured overnight, counted andsuspended in Transplant Media (Mediatech).

Rhesus macaques weighing 3-5 kg were rendered diabetic usingstreptozotocin (1250 mg/m² IV; Zanosar, Teva Parenteral Medicines,Irvine, Calif.) four weeks prior to transplantation. Diabetes wasconfirmed by intravenous glucose tolerance test (IVGTT) with a 500 mg/kgbolus of dextrose and measurement of primate C-peptide. Glucose levelswere monitored and C-peptide was measured at baseline and 10, 30, 60 and90 after injection of dextrose. Diabetes was confirmed by measurement ofelevated blood glucose levels in the absence of detectable serumC-peptide. Diabetic recipients underwent MHC-mismatched isletallotransplantation. A mean of 15,745 (±4,063) IEQ were infused via asmall midline laparotomy and cannulation of a mesenteric vein.

Blood glucose levels were measured twice daily by earstick; NPH(Novolin; Novo Nordisk, Princeton, N.J.) and glargine (Lantus;Sanofi-Aventis, Bridgewater, N.J.) insulin were administered to maintainfasting blood glucose (FBG) less than 300 mg/dL pre-transplant andfollowing graft rejection. IVGTT was performed periodicallypost-transplant to monitor graft function. Transplant recipientsunderwent weekly flow cytometric analysis to monitor T cell (CD3 V450,CD4 PerCP-Cy5.5, CD8 PerCp; BD Bioscience) and B cell (CD20 PE, BDBioscience) populations. After islet engraftment rejection was definedas FBG greater than 130 mg/dL on two consecutive days. Primary endpointwas rejection-free islet graft survival.

Transplant recipients received either 2C10R4, basiliximab (Simulect,Novartis, Basel, Switzerland) and sirolimus, or basiliximab andsirolimus alone. 2C10R4 (50 mg/kg) was administered intravenously onpost-operative day (POD) 0 and 7. Basiliximab (0.3 mg/kg) wasadministered intravenously on POD 0 and 3. Sirolimus was administeredintramuscularly daily to achieve trough levels of 5-15 ng/ml through POD120. All three animals receiving basiliximab and sirolimus alone arehistoric controls (Badell et al., J. Clin. Invest. 120:4520-312, 2010).Two of these historic controls (RQz6 and RIb7) underwent diabetesinduction by pancreatectomy and received oral sirolimus.

Treatment with the regimens described above resulted in significantlyprolonged islet graft survival (FIG. 11A) compared to controls receivingonly basiliximab induction and sirolimus maintenance therapy (FIG. 11B).Median rejection-free graft survival time for animals receiving 2C10R4is 280 days compared to 8 days for control animals (p=0.010), Table 3).Pharmacokinetic data predict that plasma 2C10R4 levels would be lessthan 1 μg/ml by POD 100. Because sirolimus was discontinued at POP120,the recipient with the longest survival (304 days) received noimmunosuppression for approximately 24 weeks prior to rejection. Noanimals treated with 2C10R4 developed clinically relevant infectiouscomplications or weight loss. These results reflect animals thatreceived the IgG4 isotype of 2C10. Two additional annuals that receivedthe IgG1 isotype of 2C10 (2C10R1) in combination with basiliximab andsirolimus achieved similarly prolonged graft survival of 220 and 162days. Given the positive results with 2C10 used as induction therapy,the next step is to assess the effects on graft survival byadministering 2C10 as maintenance therapy.

TABLE 3 Graft Recip- Survival ient Therapy IEQ/kg (days) Comment DP4A2C10R4/Basiliximab/Sirolimus 21,973 296 Rejection RAo132C10R4/Basiliximab/Sirolimus 14,388 304 Rejection RZq132C10R4/Basiliximab/Sirolimus 15,881 265 Rejection RRq132C10R4/Basiliximab/Sirolimus 20,596 163 Rejection RQz6Basiliximab/Sirolimus 12,980 8 Rejection RIb7 Basiliximab/Sirolimus10,903 8 Rejection RMc11 Basiliximab/Sirolimus 13,796 10 RejectionBlockade of the CD40/CD154 Pathway in Conjunction with the CD28/B7Pathway

Blockade of the CD40/CD154 pathway may prove useful in conjunction withother costimulation blockade agents Belatacept, a high affinity vetsionof CTLA4-Ig designed to block the CD28/B7 costimulatory pathways, hasshown efficacy in nonhuman primate models of renal and islettransplantation and in phase II and III clinical trials in renaltransplantation (Larsen, et al., Transplantation 90: 1528-35, 2010,Vincenti et al., Am. J. Transplant, 10:535-46, 2010, Adams et al., J.Immol. 174:542-50, 2005. Adams et al., Diabetes 51:205-70, 2002. Larsenet al., Am. J. Transplant. 5:443-53, 2005. Vincenti et al., N. Engl. J.Med. 358:770-81, 2005). The BENEFIT trial revealed superior renalfunction in patients treated with belatacept; however, these patientshad a higher incidence and more severe grade of biopsy-proven acuterejection (Larsen et al., Transplantation 90:1528-35, 2010, Vincenti etal., Am. J. Transplant. 10:535-46, 2010). In light of this increasedrate of acute rejection and the synergy between CD40 and B7 blockade(Larsen el al. Nature 381:434-8, 1996), we next will test the efficacyof combined 2C10 and belatacept therapy in nonhuman primate kidneytransplantation.

Example 2 Humanized Anti-CD40 Antibodies

We have developed and characterised a novel humanized Ab to CD40 calledh2C10 (humanized 2C10 antibody) that was selected as a full functionalantagonist of CD40. The binding epitopes were carefully designed toconfer unique binding properties that distinguish it from competitormolecules that either activate or deplete B cells or acts as partialagonists. The early mouse primate chimeric version of the antibody hasbeen investigated in relevant preclinical in vitro and in vivo studies,including multiple studies in nonhuman primates that demonstratepromising efficacy against preventing transplant rejection andprolonging both allo- and xenograft survival, and a favorablenonclinical safety profile. We have also completed the humanization of2C10 (h2C10), which exhibits excellent characteristics.

To produce the humanized anti-CD40 antibodies, the variable regionsequences of the murine antibody 2C10 were used to search the humanantibody database. The VH was found to be mostly related to germlineantibody sequences VH1-46, VH1-69, and VH1-3 (SEQ ID NO: 30), whereasthe VL was mostly related to germline antibody sequences VK3-11 (SEQ IDNO: 31), VK1-39, and VK6-21. The human VH1-3 and VK3-11 were chosen tobe the acceptor framework for CDR grafting because of relative highusage in human repertoire and good conservation at the criticalframework positions 3D models were built with both variable regionsafter grafting the CDRs from the murine 2C10 antibody into the humanacceptor frameworks. Six murine VH framework residues that are differentfrom the human counterparts were identified to be potentially in contactof the CDRs; M48, A67, L69, A71, K73, and N76. After modelling, threehumanized VH sequences 2C10_h1, 2C10_h2 and 2C10_h3 were designed tocontain 0, 2, and 6 murine framework residues, respectively (FIG. 13a ).Similarly, live murine VK framework residues were identified to bepotentially in contact of the CDRs; Q1, R46, W47, V58, and Y71. Aftermodeling, two humanized VL sequences 2C10_11 and 2C10_12 were designedto contain 0 and 4 murine framework residues, respectively (FIG. 13b ).

The parental murine 2C10 antibody was humanized CDR grafting. The humanantibody VH1-3 and VK3-11 germline frameworks were chosen to be theacceptor. Three VH and two VL sequences were designed and all 6humanized antibodies were produced and tested for human CD40 binding.

The 2C10-heavy-3 (2C10_h3) and 2C10_12) constructs were found to producethe best antibody with CD40 binding affinity of 0.39 within 2-fold ofthat of the murine 2C10 (0.22 nM) (Table 2). The humanized variableregions were used to construct the clinical candidate humanized antibodyas an IgG4 or a stabilized IgG4, which was cloned into the SwiMRexpression system.

High producing stable CHO cell lines were isolated by FACS and screenedby three rounds of ELISA and one round of fed-batch culture. Sevenclones were isolated that produced more than 0.8 g/L of humanized 2C10in a fed-batch culture. The best clone 3C9-I6 produced ˜1.2 g/L undernon-optimized conditions.

Construction of Antibody Expression Vectors

The humanized VH sequences were gene synthesized and cloned into vectorpFUSE-CH1g-hG2a (Invivogen) containing the constant region of human IgG2heavy chain to make expression vector LB300-302. The humanized VKsequences were gene synthesized and cloned into an expression vectorcontaining the constant region of human kappa light chain to makeexpression vector LB303-304. The heavy and light chains were downstreamof human EF1α promoter for strong and constitutive mammalian cellexpression. The chimeric 2C10 antibody was also constructed similarly byusing murine VH and VL to make expression vector LB305 and LB306,respectively. The antibody expression vectors were summarized in Table4.

TABLE 4 Antibody expression vectors Plasmid VH/VK CH/CK PromoterSelection LB300 2C10_h1 hIgG₂ CH hEF₁α Zeocin LB301 2C10_h2 hIgG₂ CHhEF₁α Zeocin LB302 2C10_h3 hIgG₂ CH hEF₁α Zeocin LB303 2C10_l1 hCK hEF₁αNeomycin LB304 2C10_l2 hCK hEF₁α Neomycin LB305 2C10_VH hIgG₂ CH hEF₁αZeocin LB306 2C10_VK hCK hEF₁α Neomycin LB308 2C10_h3 hIgG₄ CH hEF₁αZeocin LB309 2C10_h3 hIgG₄ CH hEF₁α Zeocin (S241P)

Each vector in Table 4 contains a heavy chain or light chain expressioncassette under the control of human EF1a promoter. Vectors LB3000-302,LB305 contain the constant region of human IgG2 heavy chain. VectorsLB308-309 contain the constant region of human IgG4 heavy chain. VectorsLB303-304, LB306 contain the constant region of human Kappa light chain.

Production of the Humanized IgG4 Antibody

In order to further minimize the potential effector function, thehumanized antibody with the best binding activity (2C10_h3 and 2C10_2)was converted into human IgG4 or stabilized human IgG4 (S241P). Theheavy chain variable region 2C10_h3 was first cloned into vectorpFUSE-CH1g-hG4 (Invivogen) containing the constant region of human IgG4heavy chain, before the stabilizing mutation S241P was introduced (Table4). The humanized IgG4 and IgG4 (S241P) were purified front 293F cellsafter transient transfection. The production yield was 25-35 mg/L,2-fold higher than that of the IgG2 antibodies. The IgG4 antibodyappeared to have small amount of half molecule, which was significantlyreduced in the stabilized IgG4 antibody. The DNA and amino acid sequenceof the stabilized IgG4 antibody is shown in FIG. 21.

Cloning of the Humanized IgG4 (S241P) Antibody in SwiMR ExpressionVector

SwiMR expression was developed for facile development of antibodyproduction cell lines, utilizing a switchable membrane reporter tofacilitate isolation of highly productive cells viaFluorescence-activated cell sorting (FACS). An IRES-mediated bicistronicexpression cassette of membrane-anchored GFP was placed downstream ofthe gene of interest (GOI). The IRES-GFP cassette was flanked by LoxPsites for later removal from the chromosome. The GFP expression levelwas used to mark the expression level of the GOI. Highly productivecells were isolated by FACS and then treated with Cre recombinase toremove the GPP cassette. The humanized 2C10 in the stabilized IgG4format was cloned the SwiMR expression system to make vector LB312. Theheavy and light chains were cloned in two separate expression cassettesunder control of human EF1α promoters. The IRES-GFP cassette was placeddownstream of the heavy chain sequence and was flanked by two LoxPsites. The plasmid carries a Puromycin resistant gene for mammalian cellselection and a β-lactamase gene for bacterial propagation.

Stable Selection of the CHO Cells and Isolation of the High ProducingCells

100 ml of CHOS cells (×10⁶ cells/ml. Invitrogen) were transfected with120 μg of LB312 linearized by restriction digestion of Asc I and 120 ulof Freestyle Max regent (Invitrogen). The cells were selected with 10-20ug/ml of Puromycin for 2 weeks. The GFP expression profile of the stablepool was characterized by flow cytometry. The top 1% of the cells withthe highest GFP signal was sorted out as Pool #1 containing 100,000cells. After culturing for 2 weeks, the Pool #1 was analyzed again torGFP expression by flow cytometry. The top 1% of the cells with thehighest GFP signal was sorted out again as Pool #2 containing 100,000cells. After culturing for 2 days, the Pool #2 was treated with 2 uM ofrecombinant membrane permeable DNA recombinase Cre (TAT-NLS-Cre,Excellgen). The GFP expression profile was analyzed after 1 week ofculturing. ˜10% of the cells completely lost the GFP expressionindicating successful removal of the GFP expression cassette from thechromosome. The GFP negative cells were sorted out as single cells in384-well plates. After 2 weeks, ˜800 colonies grew out from 10×384-wplates.

Additional Humanized Antibodies

Humanized antibodies were also generated using two CDR grafted VH andtwo CDR grafted VL sequences cloned into a VH1-69 and a VL1-39 humangermline framework. We made two heavy (HB1& HB2) and two light (KB1&KB2) chains in these additional experiments. The heavy and light chainsequences HP+KP serves as the positive control. The combination of theseconstructs was transiently expressed in HEK293 cells, antibody purifiedby protein-A chromatography and tested for hCD40 binding.

FIG. 14 shows amino acid changes in framework in between 2C10HP and2C10HB1, as well as 2C10HB2 constructs. FIG. 15 shows the sequences ofheavy chain and light chain variable regions for humanized 2C10antibodies. The heavy chain and light chain variable regions include2C10HP, 2C10HB1, 2C10HB2, 2C10KP, 2C10KB1, and 2C10KB2. Therefore, incertain embodiments an anti-CD40 antibody may include any of thefollowing 2C10H-K combinations:

-   -   1. 2C10HP+2C10KP    -   2. 2C10HB1+2C10KB1    -   3. 2C10HB1+2C10KB2    -   4. 2C10HB1+2C10KP    -   5. 2C10HB2+2C10KB2    -   6. 2C10HB2+2C10KB1    -   7. 2C10HB2+2C10KP    -   8. 2C10HP+2C10KB1    -   9. 2C10HP+2C10KB2        In Vitro Binding of CD40 with Purified Antibodies

Humanized antibodies and the chimeric antibody were purified aftertransient transection of 100 or 200 ml of 293F cells. The antibodieswere purified with a Protein A column from the conditioned mediaharvested 4 days after transfection.

Determination of CD40 Binding Kinetics

CD40 binding kinetics was determined on Forte Bio (contracted to AragenBioscience). The purified CD40 was biotinylated and immobilized onStrepiavidin biosensors.

Bioproduction for In Vivo Study

After transfection of CHO cells and selection of stably transfectedcells, the antibody was purified by Protein A column, and followed bybuffer exchange (20 mM Sodium Citrate, 50 mM NaCl, 5% Maltose, pH 6.0)and 0.2 μm filtration. The Pool #1 was used to setup 25 L wave bagculture in CD FortiCHO media (Invitrogen). The culture was fed threetimes on day 3, 5, and 7 with 10% CD Efficient Feed C (Invitrogen). Thefinal yield of purified antibody was 1.6 g in total. The antibody wascharacterized by SDS-PAGE and SEC-HPLC analysis, and was 99.4% pure asmonomeric antibody.

Cell Line Development

The single cell colonies were screened by 3 rounds of ELISA and 1 roundof fed-batch production. The cells were kept in CD FortiCHO mediathroughout the screening process. All colonies from 384-well plates werepicked into 96-well plates. 1.2 μl of culture media from each well wereused to screen for antibody in ELISA plates coated with anti-human Fcantibody. The top 240 clones were expanded into 10×24-well plates. Afterculturing for 5 days, 1.2 μl of culture media was screened again forantibody level, the top 60 clones were expanded into 10×6-well platesand cultured in shaking incubator. After culturing for 5 days, the6-well plates were duplicated by passaging the cells 1:10 into a new setof 6-well plates. The cultures in the original set of the 6-well plateswere allowed to grow to extinction, followed by determination ofantibody level by ELISA. The top 24 clones in the duplicated set of6-well plates were expanded into 30 ml culture in 125 ml shake flasks.The clones were subjected to 30 ml fed-batch production. The feedingstrategy was 7.5% of Ex-Cell Advanced CHO Feed 1 (Sigma) on day 3, 5, 7,9, and 11. The top clone 3C9-I6 exhibited production titer of ˜1.2 g/L.

In Vitro Pharmacology of Primate Chimeric 2C10 and Humanized 2C10

We identified the following important in vitro pharmacologicalattributes for our lead candidate:

-   -   Suppression of B-cell activation induced by CD154-CD40        engagement    -   No direct activation of B cells    -   High affinity antagonist of CD40 (e.g., K_(d) is about 10⁻¹⁶M or        lower, about 10⁻¹⁰ M to about 10⁻⁹M, or as described herein)

Through a novel immunization approach and an extensive in vitroscreening approach, we have identified an anti-CD40 antibody that meetsthese criteria and represents a non-depleting/non-activating antagonistantibody to human CD40.

In vitro and in vivo studies have confirmed that the humanized formretains excellent properties.

As described in the previous section, the 2C10 mAb was humanized by CDRgrafting into a human heavy and light chain frameworks. To maintain theproperties of the original 2C10 mAb, the humanized 2C10 constructs werescreened by Biacore for affinity 10 human CD40. All three top humanized2C10 antibodies exhibited only slight reduction in affinity byapproximately two-fold, relative to the parent 2C10 mAb (Table 5). Mostimportantly they all maintained the exceptional slow off rate of theparent 2C10 mAb. Among these antibodies, Clone 2.189.2 which exhibitedthe highest affinity at 390 pM; was selected as the lead humanized mAb(h2C10).

TABLE 5 CD40 Receptor Binding Kinetics of Humanized Versions of 2C10 mAbK_(D) (M) K_(on) (1/Ms) K_(off) (l/s) Rmax Full X{circumflex over ( )}2Full R{circumflex over ( )}2 2C10 2.22E−10 1.48E+05 6.01E−05 0.31240.284838 0.993451 2.189.1 5.11E−10 1.98E+05 1.85E−04 0.3917 0.4906590.991111 2.189.2 3.90E−10 1.79E+05 1.28E−04 0.3946 0.401784 0.9916972.191.1 5.61E−10 1.84E+05 1.88E−04 0.3924 0.402934 0.992955

Comparing the binding kinetics of humanized 2C10 to competitors, theoverall affinity of h2C10 remains substantially better than thecompetitors that have affinities in the nanomolar range. We alsocompared binding affinity of h2C10 between human CD40 and CD40 fromthose of nonhuman primate species used in preclinical evaluations. Asshown in FIG. 16, h2C10 has comparable affinity for CD40 across theseprimate species.

In Vivo Characterization of Primate Chimeric and Humanized 2C10

The in vivo pharmacodynamics, pharmacokinetics and exploratory safetyassessments of 2C10 were conducted in rhesus monkeys using the primatechimeric construct of 2C10 and the clinical candidate humanized h2C10antibody. After selection of the lead humanized version of 2C10 (mAb2.189.2: h2C10) based on in vitro binding kinetics, we advanced h2C10into a PK/PD study in rhesus monkeys to characterize its additionalproperties. The data generated in these studies, which cover a broadrange of critical experimental endpoints, clearly establish theexcellent properties of h2C10.

Studies have been completed in rhesus monkeys examining PD, PK, andsafety endpoints. Critical elements of the study designs including keyendpoints and objectives are summarized in Table 6 (Pharmacodynamic(PD), Pharmacokinetic (PK) and Safety Studies of 2C10 in RhesusMonkeys). The key outcomes and relevant comparative assessments on thekey experimental endpoints included:

-   -   Effect on B and T lymphocyte number in blood    -   Effect on humoral immune response to the T cell-dependent        antigen Keyhole Limpet Hemocyanin (KLH)    -   CD40 receptor occupancy on blood B cells (PD)    -   Pharmacokinetics (PK)    -   Immunogenicity as assessed by formation of anti-drug antibodies        (ADA)    -   Exploratory toxicology include CBC, serum chemistry and complete        necropsy

TABLE 6 PD, PK and Safety Studies of 2C10 in Rhesus Monkeys Expt. KeyTest Group Test Doses/ Follow-Up # Objective(s) Versions Size RegimenPeriod Key Endpoints 1 Compare Primate 3 Two Doses; 56 Days CBC IgG1 andchimeric 50 mk/kg, Lymphocyte Subsets IgG4 forms, 2C10 IV on DaysPrimary TDAR and 0 and 7; (anti-KLH) Competitor Saline- 3A8 Control 2Dose- Primate 3 One Dose; 56 Days CBC Response chimeric 5, 10, 25, 50Lymphocyte Subsets Evaluation 2C10 mg/kg, IV; Detailed B Cell IgG4Irrelevant Subsets TDAR IgG (primary and Control recall response to KLH)3 Safety Primate 2 Two Doses; 14 Days CBC Assessment/ chimeric 25 mg/kg,after Serum Chemistry Exploratory 2C10 IV on Day second LymphocyteSubsets Toxicology IgG4 0 and 14; dose (28 Necropsy Gross & HistoricalDays in Microscopic Pathology Control Total) 4 PK, PD, Humanized 3 OnDose; 28 Days Receptor Occupancy Safety 2C10 10, 25 CBC Serum ChemistryIgG4 mg/kg, IV; Lymphocyte Subsets Saline Primary TDAR Controlanti-KLH)PK ADA

The study results are summarized in the following section, h2C10 may beused for treatment of conditions in which selective blockade of CD40receptor activation in the absence of B cell depletion is expected toprovide therapeutic benefit.

Effects on T Cell-Mediated Immunity

To prove in vivo that h2C10 blocks T-cell dependent antibody responses(TDAR) in vivo, monkeys were immunized with KLH 6 hours after theadministration of h2C10. Antibody titers against KLH were measuredweekly thereafter.

Doses of 10 and 25 mg/kg h2C10 were given to monkeys followed by a KLHchallenge. Both IgG and IgM anti-KLH titers were determined weekly outto Day 28 after treatment. FIG. 17 shows that the humanized version of2C10 achieved complete inhibition of the KLH antibody response at thehighest test dose, and in most cases at the 10 mg/kg dose level. BothIgM and IgG responses were prevented.

As goals in the development of an antagonist CD40 antibody includedminimizing the depletion of targeted. CD40+ cells, we analysed effectson lymphocyte subpopulations, especially the effect on B cells.

The treatment of monkeys with 10 or 25 mg kg of the humanized form of2C10 (h2C10) also had no appreciable depleting effect on B cells eventhough the CD40 target is fully saturated by the antibody at theseconcentrations (see subsequent Section Receptor Occupancy).

Despite saturation of 2C10 binding sites on B cells lasting until thelast day measured (Day 28), all monkeys that received either dose ofh2C10 maintained normal B and T lymphocyte subsets (FIG. 18). Theseresults demonstrate that injection with a dose as low as 10 mg/kg 2C10can persistently bind CD40, its therapeutic target on B cells (andpresumably monocytes and other antigen presenting cells) without causingundesired depletion of B cells in monkeys.

The demonstration that 2C10 does not deplete B cells suggests a clearadvantage over competitors in addition to 3A8 and Chi220.

Pharmacodynamics

CD40 Receptor Occupancy

CD40 target, engagement and occupancy by the primate chimeric andhumanized versions of 2C10 was determined by measuring the availablebinding sites for 2C10 to CD40 on CD20+ B cells by flow cytometry usingfluorescently labeled 2C10 and a labeled, non-competing anti-CD40antibody. Blood samples were collected on multiple days from controlmonkeys and monkeys treated with either primate chimeric IgG4 orhumanized 2C10 and analyzed by FACS. The degree of target engagement (%receptor occupancy) was calculated directly from the mean fluorescentintensity recordings.

Humanized 2C10 antibodies were administered intravenously at singledoses of 10 and 25 mg/kg. Surface CD40 on B cells was completelysaturated by Day 3, and the effect persisted until the last day measured(Day 28) in all monkeys that received either dose of h2C10.Representative data from the flow cytometry analysts of blood collectedfrom monkeys 28 days alter treatment with humanized 2C10 is shown FIG.19.

These results demonstrate that a single injection of h2C10 at a dose aslow as 10 mg/kg can fully saturate CD40 receptors on B cells for atleast 28 days.

Pharmacokinetics and Anti-Drug Antibody Response

To establish a clear link between the pharmacodynamic effects of 2C10based on CD40 receptor occupancy and the pharmacokinetics of 2C10 plasmaconcentrations of the 2C10 were measured in plasma from the same bloodsamples in which receptor occupancy had been determined. The plasmaconcentration analysis also enabled characterization of thepharmacokinetic profile of 2C10, most importantly its persistence inplasma as determined by its half-life. This determination providesguidance for the frequency of dosing that will be needed to maintaineffective therapeutic concentrations.

The mean serum concentrations determined in monkeys treated with either10 or 25 mg/kg humanized 2C10 are plotted in FIG. 20. These datademonstrate that the animals were exposed to 2C10 for the entireduration of the study, and that humanized 2C10 has a half-life inmonkeys of approximately 15 days (ranging from 9-20 days). Thishalf-life is in the range of that expected for a therapeutic antibody inprimates, and should support a relatively infrequent dosing schedule inclinical investigations (e.g., no more than once every two weeks).Further modeling of the complete dataset will enable a robust estimateof the dose and frequency required to sustain effective antibodyconcentrations in initial clinical studies of h2C10.

Another assessment of humanized 2C10 was the potential development ofantibodies against h2C10 (ADA). This can occur when biologies ofepitopes from one species is administered to a different species (eg.Humanized mAb to primates) resulting in rapid clearance of the drug fromplasma. In this study there was no evidence from the time course profilethat antibodies against h2C10 were generated, as no animal exhibitedmeasurable anti-2C10 titers during the study.

Preliminary Safety Evaluation

The experiments described in this section were conducted to determinepotential adverse consequences of h2C10 on the immune system and foroff-target effects. For biological therapies, these assessments areamong the most critical for evaluating safety prior to human exposure tothe drug, and developing risk mitigation plans for clinical testing. Theabsence of any unexpected or undesired outcomes on immune function orpathology in monkeys is viewed favorably for the overall safetyassessment of h2C10. In addition, routine tests for alterations inhematological parameters, including platelet counts, and serum chemistryparameters in monkeys treated with 2C10 were conducted on several dayspost-dosing, and were unaffected by treatment with chimeric andhumanized 2C10. Two animals that were treated twice with 25 mg/kgprimate chimeric 2C10 were evaluated for gross and microscopic evidenceof treatment related pathology; no treatment-related pathologicalchanges were observed. In addition, to rule out thromoboemoliccomplications, all tissues were examined by special stains for fibrindeposition. No evidence of subclinical clotting abnormalities wasdetected. Importantly, relatively high doses were tested in the studiesthat appreciably exceeded the doses required to full occupy CD40receptors and therefore approach the dose levels that will be tested inpivotal toxicology studies conducted during the IND-enabling phase.These preliminary safety evaluations show absence of any safety concernsfor h2C10.

These combined data demonstrate in a relevant primate model that 2C10has the desirable pharmacodynamic, pharmacokinetic and safety attributesfor a monoclonal antibody intended to treat patients, where specificinhibition of CD40 actuation is desired without causing unwantedactivation or depletion of CD40+ targets cells and without evidence ofoff-target toxicity.

While specific aspects of the invention have been described andillustrated, such aspects should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims. All publications and patentapplications if each individual publication or patent application werespecifically and individually indicated to be incorporated by referencefor all purposes. Although the foregoing invention has been described insome detail by way of illustration and example for purposes of clarityof understanding, it will be readily apparent to one of ordinary skillin the art in light of the teachings of this invention that certainchanges and modifications can be made thereto without departing from thespirit or scope of the appended claims.

What is claimed is:
 1. A humanized anti-CD40 antibody or antigen-bindingfragment thereof comprising a heavy chain variable region comprising anamino acid sequence with at least 80% sequence identity to the aminoacid sequence set forth in any one of SEQ ID NOs: 19, 20, 21, 24, 25,and 26 and a light chain variable region comprising an amino acidsequence with at least 80% sequence identity to the amino acid sequenceset forth in any one of SEQ ID NOs: 22, 23, 27, 28 and 29, wherein theheavy chain variable region comprises a CDR1, CDR2, and CDR3 with theamino acid sequences set forth in SEQ ID NOs: 13, 14, and 15,respectively, and wherein the light chain variable region comprises aCDR1, CDR2, and CDR3 with the amino acid sequences set forth in SEQ IDNOs: 16, 17, and 18, respectively.
 2. The antibody or antigen-bindingfragment thereof of claim 1, wherein the amino acid sequence of theheavy chain variable region has at least 90% sequence identity to theamino acid sequence set forth in any one of SEQ ID NOs: 19, 20, 21, 24,25, and 26, and the amino acid sequence of the light chain variableregion has at least 90% sequence identity to the amino acid sequence setforth in any one of SEQ ID NOs: 22, 23, 27, 28 and
 29. 3. The antibodyor antigen-binding fragment thereof of claim 2, wherein the amino acidsequence of the heavy chain variable region comprising an amino acidsequence has at least 95% identity to the amino acid sequences set forthin any one of SEQ ID NOs: 19, 20, 21, 24, 25, and 26, and the amino acidsequence of the light chain variable region has at least 95% sequenceidentity to the amino acid sequences set forth in any one of SEQ ID NOs:22, 23, 27, 28 and
 29. 4. The antibody or antigen-binding fragmentthereof of claim 3, wherein the amino acid sequence of the heavy chainvariable region comprises the amino acid sequence set forth in any oneof SEQ ID NOs: 19, 20, 21, 24, 25, and 26, and the amino acid sequenceof the light chain variable region comprises the amino acid sequence setforth in any one of SEQ ID NOs: 22, 23, 27, 28 and 29, wherein theantibody or antigen-binding fragment thereof does not comprise the heavychain variable region comprising the amino acid sequence set forth inSEQ ID NO: 21 and the light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:
 23. 5. The antibody orantigen-binding fragment thereof of claim 1, wherein: a) thedissociation constant (KD) of the antibody or antigen-binding fragmentthereof is less than about 1×10⁻⁹ M; b) the antibody or antigen-bindingfragment thereof is selected from the group consisting of: (a) a wholeimmunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) anF(ab′)₂; and (e) a disulfide linked Fv; c) the antibody orantigen-binding fragment thereof comprises at least one constant domainselected from the group consisting of: a) an IgG constant domain; and(b) an IgA constant domain; d) the antibody or antigen-binding fragmentthereof comprises at least one human constant domain; e) the antibody orantigen-binding fragment thereof binds to CD40 extracellular domain; f)the CD40 is human or rhesus CD40; g) the antibody or antigen-bindingfragment thereof blocks B lymphocyte activation by CD154-expressingJurkat cells in vitro; and/or h) the antibody or antigen-bindingfragment thereof inhibits B lymphocyte CD23, CD80, or CD86 expression.6. A composition comprising the antibody or antigen-binding fragmentthereof of claim 1 and a pharmaceutically acceptable carrier.
 7. Amethod of suppressing the immune system in a subject comprisingadministering to the subject an effective amount of the antibody orantigen-binding fragment thereof of claim
 1. 8. A method of treatingtransplant rejection or increasing the duration of time beforetransplant rejection occurs in a subject in need thereof comprisingadministering to the subject an effective amount of the antibody orantigen-binding fragment of claim
 1. 9. The method of claim 8, whereinthe subject has received, or is in need of, an organ or tissuetransplantation.
 10. The method of claim 9, wherein: a) the organ isselected from the group consisting of heart, kidney, lung, liver,pancreas, intestine, and thymus, or a portion thereof; or b) the tissueis bone, tendon, cornea, skin, heart valve, vein, or bone marrow. 11.The method of claim 8, wherein the antibody or antigen-binding fragmentthereof is administered prior to the transplantation.
 12. The method ofclaim 8, wherein the antibody or antigen-binding fragment thereof isadministered for at least one month following the transplantation. 13.The method of claim 12, wherein the antibody or antigen-binding fragmentthereof is administered for at least six months following thetransplantation.
 14. A method of treating graft-versus-host disease in asubject in need thereof comprising administering to the subject atherapeutically effective amount of the antibody or antigen-bindingfragment thereof of claim
 1. 15. A method of blocking the ability ofCD40 to interact with CD154 in a subject comprising administering to thesubject an effective amount of the antibody or antigen-binding fragmentthereof of claim
 1. 16. The method of claim 15, wherein the subject hasan autoimmune disorder associated with or caused by the presence of anautoantibody.
 17. The method of claim 15, wherein the subject has adisorder selected from the group consisting of systemic lupuserythematosus (SLE), CREST syndrome (calcinosis, Raynaud's syndrome,esophageal dysmotility, sclerodactyl, and telangiectasia), opsoclonus,inflammatory myopathy, systemic scleroderma, primary biliary cirrhosis,celiac disease, dermatitis herpetiformis, Miller-Fisher Syndrome, acutemotor axonal neuropathy (AMAN), multifocal motor neuropathy withconduction block, autoimmune hepatitis, antiphospholipid syndrome,Wegener's granulomatosis, microscopic polyangiitis, Churg-Strausssyndrome, rheumatoid arthritis, chronic autoimmune hepatitis,scleromyositis, myasthenia gravis, Lambert-Eaton myasthenic syndrome,Hashimoto's thyroiditis, Graves' disease, Paraneoplastic cerebellardegeneration, Stiff person syndrome, limbic encephalitis, IsaacsSyndrome, Sydenham's chorea, pediatric autoimmune neuropsychiatricdisease associated with Streptococcus (PANDAS), encephalitis, diabetesmellitus type 1, neuromyelitis optica, pernicious anemia, Addison'sdisease, psoriasis, inflammatory bowel disease, psoriatic arthritis,Sjögren's yndrome, lupus erythematosus, multiple sclerosis, reactivearthritis, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupoidhepatitis, atherosclerosis, presenile dementia, demyelinating diseases,subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler'ssyndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenicpurpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopeciaarcata, pemphigoid, scleroderma, progressive systemic sclerosis,adult-onset diabetes mellitus, male and female autoimmune infertility,ankylosing spondolytis, ulcerative colitis, Crohn's disease, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, juvenile onset rheumatoid arthritis, glomerulonephritis,atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas'disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion,anti-phospholipid syndrome, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, autoimmune chronic activehepatitis, bird-fancier's lung, allergic disease, allergicencephalomyelitis, toxic epidermal necrolysis, alopecia, Alport'ssyndrome, alveolitis, allergic alveolitis, fibrosing alveolitis,interstitial lung disease, erythema nodosum, pyoderma gangrenosum,transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis,Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis,schistosomiasis, giant cell arteritis, ascariasis, aspergillosis,Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet'sdisease, Caplan's syndrome, Kawasaki's disease, dengue, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome,Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochroniccyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura,graft versus host disease, transplantation rejection, humanimmunodeficiency virus infection, echovirus infection, cardiomyopathy,Alzheimer's disease, parvovirus infection, rubella virus infection, postvaccination syndromes, congenital rubella infection, Hodgkin's and nonHodgkin's lymphoma, renal cell carcinoma, multiple myeloma,Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma,cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barr virusinfection, mumps, Evan's syndrome, and autoimmune gonadal failure. 18.The method of claim 7, wherein the subject is a human.
 19. The method ofclaim 7, wherein the antibody or antigen-binding fragment thereof isadministered via parenteral, intravenous, subcutaneous, intramuscular,transdermal, oral, topical, intrathecal, or local routes.
 20. The methodof claim 7, wherein the method further comprises administering animmunosuppressant within six months of administering the antibody orantigen-binding fragment thereof.
 21. The method of claim 20, whereinthe immunosuppressant is selected from the group consisting of acalcineurin inhibitor selected from the group consisting, of cyclosporinA and cyclosporine G, tacrolimus, an mTor inhibitor, fingolimod,myriocin, alemtuzumab, rituximab, an anti-CD4 monoclonal antibody, ananti-LEA-1 monoclonal antibody, an anti-LEA-3 monoclonal antibody, ananti-CD45 antibody, an anti-CD19 antibody, monabatacept, belatacept,indolyl-ASC; azathioprine, lymphocyte immune globulin and anti thymocyteglobulin, mycophenolate mofetil, mycophenolate sodium, daclizumab,basiliximab, cyclophosphamide, prednisone, prednisolone, leflunomide,FK778, FK779, 15-deoxyspergualin, busulfan, fludarabine, methotrexate,6-mercaptopurine, 15-deoxyspergualin, LF15-0195, busulfan, fludarabine,methotrexate, 6-mercaptopurine, 15-deoxyspergualin, LF15-0195, bredinin,brequinar, and muromonab-CD3.
 22. The method of claim 21, wherein thecalcineurin inhibitor is cyclosporin A or cyclosporine G.
 23. The methodof claim 21, wherein the anti-CD45 antibody is an anti-CD45RB antibody.24. The method of claim 21, wherein the immunosuppressant is belatacept.25. The method of claim 21, wherein the antibody or antigen-bindingfragment thereof and the immunosuppressant are administered within onemonth of each other.
 26. The method of claim 25, wherein the antibody orantigen-binding fragment thereof and the immunosuppressant areadministered within one week of each other.